Bacteria (Living Resources for Biotechnology)

This volume is a unique international compilation for biotechnologists of data on the source and use of bacterial cells. The volume provides details of the location and scope of major culture collections around the world holding bacteria; information is given on how to access their data, administration and safety, identification, culture, preservation, patents, specialist services and international organisations. The authors are international authorities who have combined with the resource centres to provide a source book for microbiologists in industry, research establishments and universities.

An international initiative by the World Federation for Culture Collections, with financial support from UNESCO LIVING RESOURCES FOR BIOTECHNOLOGY Editorial Board: A. Doyle D. L. Hawksworth L. R. Hill B. E. Kirsop (Senior Editor) K. Komagata R. E. Stevenson

Bacteria

Titles in the series Animal cells Bacteria Filamentous fungi Yeasts

LIVING RESOURCES FOR BIOTECHNOLOGY

Bacteria Edited by

L. R. Hill and B. E. Kirsop

The right of the University of Cambridge to print and sell all manner of books was granted by Henry VIII in 1534. The University has printed and published continuously since 1584.

CAMBRIDGE UNIVERSITY PRESS Cambridge New York Port Chester Melbourne Sydney

CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, Sao Paulo Cambridge University Press The Edinburgh Building, Cambridge CB2 2RU, UK Published in the United States of America by Cambridge University Press, New York www.cambridge.org Information on this title: www.cambridge.org/9780521352246 © Cambridge University Press 1991 This publication is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 1991 A catalogue recordfor this publication is available from the British Library Library of Congress Cataloguing in Publication data Bacteria/edited by L. R. Hill and B. E. Kirsop. p. cm. - (Living resources for biotechnology) Includes bibliographical references Includes index. 1. Bacteria - Type specimens - Catalogs and collections. 2. Bacteria - Type specimens - Catalogs and collections - Directories. 3. Biotechnological microorganisms - Catalogs and collections - Directories. I. Hill, L. R. (Leslie Rowland), 1935II. Kirsop, B. E. III. Series. [DNLM: 1. Bacteria. 2. Biotechnology. QW 4 B1303] QR64.5.B33 1990 660'.62-dc20 90-2024 CIP ISBN-13 978-0-521-35224-6 hardback ISBN-10 0-521-35224-X hardback Transferred to digital printing 2005

CONTENTS

Contributors Series introduction

ix xi

xiii Resource centres L. R. Hill, K. Komagata and R. L. Gherna 1 1 Nature of the resource 2 Biotechnological applications 3 Types of resource centres 20 Future development of resource centres 21 Reference Information resources M. I. Krichevsky and H. Sugawara 22 22 Introduction 23 Information needs 28 Information resources 46 Access to data resources 49 Administration and safety L. R. Hill and R. L. Gherna 49 Administration 54 Safety 61 References Culture and maintenance L. R. Hill, M. Kocur and K. A. 62 Malik 62 Culture 64 Maintenance 77 Quality control 78 Records 78 Security 78 References Preface

1 1.1 1.2 1.3 1.4 1.5 2. 2.1 2.2 2.3 2.4 3 3.1 3.2 3.3 4 4.1 4.2 4.3 4.4 4.5 4.6

vn

viii

Contents 5 5.1 5.2 5.3 5.4 5.5

Identification L. R. Hill, K. A. Malik and K. Komagata Introduction Practical aspects of identification Chemotaxonomy Nucleic acids Selected references for identification

81 81 86 89 91 93

6 Patent protection for biotechnological inventions LJ. Bousfield 6.1 Introduction 6.2 Basis of the patent system 6.3 Kinds of biotechnological inventions 6.4 Patentability of biotechnological inventions 6.5 Practical considerations 6.6 Further reading

95 95 95 97 98 110 131

7 7.1 7.2 7.3 7.4 7.5 7.6

134 134 134 141 142 143 144

Culture collection services D. Allsopp and F. P. Simione Introduction Types of services Workshops and training Publications, catalogues and publicity material Fees and charges Suggested reading

8 Organisation of resource centres B. E. Kirsop and E. J. DaSilva 8.1 Introduction 8.2 International organisation 8.3 Regional organisation 8.4 National federations/committees 8.5 Future developments Index

146 146 146 156 158 159 162

CONTRIBUTORS

Allsopp, D. CAB International Mycological Institute, Ferry Lane, Kew, Surrey TW9 3AF, UK (Chapter 7) Bousfield, I. J. National Collections of Industrial and Marine Bacteria Ltd, 23 St. Machar Drive, Aberdeen AB2 1RY, UK (Chapter 6) DaSilva, E. J. United Nations Educational Scientific and Cultural Organisation, Division of Scientific Research and Higher Education, 7 Place de Fontenoy, F-75700 Paris, France (Chapter 8) Gherna, R. L. American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA (Chapters 1, 3) Hill L. R. National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK (Chapters 1, 3, 4, 5) Kirsop, B. E. Microbial Strain Data Network, Institute of Biotechnology, Cambridge University, 307 Huntingdon Road, Cambridge CB3 OJX, UK (Chapter 8) Kocur, M. Czechoslovak Collection of Microorganisms, Masaryk University, Jostova 10, Brno 66243, Czechoslovakia (Chapter 4) Komagata, K. Hakusan 1-5-10-503, Bunkyo-ku, Tokyo 114, lapan (Chapters 1, 5) Krichevsky, M. I. Microbial Systematics Division, National ix

Contributors

Institute of Dental Research, Bethesda, Maryland 20892, USA (Chapter 2) Malik, K. A. Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 1, D-3300 Braunschweig, Federal Republic of Germany (Chapters 4, 5) Simione, F. P. American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 USA (Chapter 7) Sugawara, H. World Data Center for Collections of Microorganisms, Life Science Research and Information Section, RIKEN, Wako, Saitama 351-01, Japan (Chapter 2)

SERIES INTRODUCTION

The rapid advances taking place in biotechnology have introduced large numbers of scientists and engineers to the need for handling microorganisms, often for the first time. Questions are frequently raised concerning sources of cultures, location of strains with particular properties, requirements for handling the cultures, preservation and identification methods, regulations for shipping, or the deposit of strains for patent purposes. For those in industry, research institutes or universities with little experience in these areas, resolving such difficulties may seem overwhelming. The purpose of the World Federation for Culture Collections' (WFCC) series, Living Resources for Biotechnology, is to provide answers to these questions. Living Resources for Biotechnology is a series of practical books that provide primary data and guides to sources for further information on matters relating to the location and use of different kinds of biological material of interest to biotechnologists. A deliberate decision was taken to produce separate volumes for each group of microorganism rather than a combined compendium, since our enquiries suggested that inexpensive specialised books would be of more general value than a larger volume containing information irrelevant to workers with interests in one particular type of organism. As a result each volume contains specialised information together with material on general matters (information centres, patents, consumer services, the international coordination of culture collection activities) that is common to each. The WFCC is an international organisation concerned with the establishment of microbial resource centres and the promotion of their activities. In addition to its primary role of coordinating the work of culture collections through the world, the committees of the WFCC are xi

xii

Series introduction

active in a number of areas of particular relevance to biotechnology, such as patents, microbial information centres, postal and quarantine regulations, educational and conservation matters (see Chapter 8). The Education Committee of the WFCC proposed the preparation of the current volumes. The WFCC is concerned that this series of books is of value to biotechnologists internationally, and the authors have been drawn from specialists throughout the world. The close collaboration that exists between culture collections in every continent has made the compilation of material for the books a simple and pleasurable process, since the authors and contributors are for the most part colleagues. The Federation hopes that the result of their labours has produced valuable source books that will not only accelerate the progress of biotechnology, but will also increase communication between culture collections and their users to the benefit of both. Barbara Kirsop President, World Federation for Culture Collections

PREFACE

Bacteria have a daily impact upon human activities. The emergence of the new biotechnology has increased the awareness by scientists of the long recognised need for reliable, permanent, culture collections which safe-keep viable exemplars of the many known bacterial species and varieties. There is now an increased awareness too that what is in fact conserved in service collections represents but a small part of the bacterial gene pool. Outside the recognised and long-established 'servicesupply culture collections' there are many other centres whose holdings of cultures add to overall microbial, living resources available to scientists. There is an emphasis in this book on what defines a useful microbial resource: the cultures themselves, their documentation, and increasingly wide knowledge of their existence. Today we are also in an age of developing information technology. Progress here enhances the existing resources, making it increasingly easy for individual scientists to access the great body of technical information associated with holdings of cultures. An additional benefit from the use of information technology to improve wider access to known information is to bring more clearly into focus gaps in our present knowledge and shortfalls in the presently conserved ranges of organisms available. This book is an introduction to these resources, to culture collections, their holdings, and to the ways and means scientists responsible for their upkeep are exploiting information technology in the service of science. Hopefully, it will act as a stimulus to both research scientists and those engaged even in focused applied work. Reality dictates that often the distinction between research and applied science is blurred, but the extremes of each have need for authenticated, documented xiii

xiv

Preface

exemplars of the known microbial gene pool. As new elements are discovered or found to have new applications, it can only benefit science if positive steps are made to ensure preservation for the future. Existing living resources for biotechnology are already extensive, but in no way exhaustive. I am very grateful for the assistance of colleagues and contributors to the various chapters. We have tried to present as international a perspective as possible, a task that inevitably will be incomplete. L. R. Hill London Curator, National Collection of Type Cultures

1 Resource Centres L. R. HILL, K. KOMAGATA and R. L. GHERNA

1.1

Nature of the resource

Living and authenticated cultures of bacteria are essential in virtually all practical applications of bacteriology, whether for routine work or for research. Such cultures are needed as controls, sources of special products, indicators of particular reactions or interactions; as representatives of their kind, or species (important for comparative identifications); as standards. Far from detracting from these 'classical' needs, the advent of the biotechnology of today has simply added more user needs, often, however, with a demand for greater amounts of information about the cultures. There are three principal elements that convert a culture, or collection of cultures, into a 'resource'. First is the culture itself, successfully preserved in a viable state. If viability cannot be maintained, either technically or simply through default, then only the information that may have been published is a resource: to exploit this limited resource a new isolate has to be obtained. Second is the information recorded about the culture. This includes such documentation as the history of the culture, original source, and also a record of the properties of the culture. The information may be little or exhaustive, but the preservation of cultures without information means they form a substantially less useful resource. Third is the availability of the culture, to enable its further application. However well preserved and well documented, a culture cannot be regarded as a 'resource' if it is not available: at best, it is a resource solely for its owner. Availability may, of course, be subject to restrictions or conditions; for example, hazardous bacteria (see Chapter 4) or patent strains (see Chapter 6) may not be universally available. 1

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L. R. Hill, K. Komagata and R. L. Gherna

Many cultures are resource materials precisely because they have particular, documented applications or properties; others may be representatives of new species, with no immediate practical utility. However, this does not exclude as a valuable resource the conservation of a selection of cultures for no immediately known purpose. We cannot guess today what materials may be useful in the future, nor for what purpose; we cannot predict what changes will occur in the general environment or special habitats, nor what effects such changes may ultimately have on bacterial genomes or extra-chromosomal elements. Thus, it is important to conserve for posterity exemplars of the bacterial spectrum of today and to include some about which our knowledge may be very limited and others of no known present application. Living cultures that are documented and available are, then, the basic resource. Laboratories which make a deliberate effort to maintain the basic resource qualify as resource centres. These will range from small collections, where conservation is a secondary activity of the laboratory, to larger, perhaps specialised, collections with an increased effort to maintain the resource successfully, and finally to the nationally and internationally recognised service-supply culture collections where conservation is the primary function. Conservation of the genetic resources of today for posterity involves active accessioning policies, successful preservation of cultures, quality control procedures in addition to complete documentation and capacity to supply cultures to the scientific community. Resource centres provide, to varying degrees, a further utility: their scientific expertise relative to the cultures under their curatorial responsibility. Even in the large service-supply culture collections, where the range of bacteria maintained is so wide it is impossible for in-house staff to be expert with all the species, the availability of scientific expertise is itself a valuable resource. This expertise may be direct, deriving from, for example, pertinent research programmes of the resource centre itself, or indirect, by knowledge of who elsewhere is likely to provide the answer to a particular query. 1.2

Biotechnological applications There are many early and continuing biotechnological applications useful to mankind which use bacteria as agents in particular processes - some natural, some induced - or as sources of useful products. Modern biotechnology, however, concerns genetic engineer-

Resource centres

3

ing, manipulation of bacterial DNA to accommodate foreign DNA and thereby to change the properties of the bacterium in a way beneficial to man. This is not the place even to list the variety of applications actually or potentially carried out by altered bacteria in this rapidly expanding activity called 'biotechnology'. It is pertinent to note, however, that modern biotechnology demands high levels of scientific expertise to carry out manipulations, and technical expertise to transform a laboratory construct into an economic industrial process. Each successful application represents a significant effort and the bacteriological end-product, namely the successfully manipulated bacterium, is a valuable commodity. Conservation of this commodity then becomes very important and deposit (for private safe-keeping, as open deposits, or as patent deposits) in a reliable resource centre becomes essential. The large service-supply culture collections have responded positively to this new biotechnological need. Already biotechnology has made another impact on resource centres. The information content of collection catalogues, usually arranged systematically by genus and species names (which in themselves may convey a significant quantity of information on properties), giving the isolation-cultural 'pedigree' of cultures and, if appropriate, some special properties or uses, is adequate for many purposes. Modern biotechnology, however, places a demand for more detailed information on properties, often of a specialist kind. Where the resource centres have relevant information, additional to that appearing in catalogues, they are responding by constructing computer databases, and by means of computer networks are providing direct or indirect access to these (see Chapter 2). 1.3

Types of resource centres

1.3.1

Service-supply culture collections

Service-supply collections exist primarily to maintain and supply on demand catalogued, authenticated cultures. They are the primary sources of comparable materials, permitting workers in different times and/or places to work with the same materials, thus making their work to a degree standardised and comparable with others. Their utility to the scientific community has been long recognised and some of the largest service-supply collections were founded in the first quarter of this century. The American Type Culture Collection (ATCC, founded 1925) houses collections relevant to a wide range of microbiological subdisciplines and not solely bacteriology. Elsewhere, long

4

L. R. Hill, K. Komagata and R. L. Gherna

established and internationally well known service collections are specialised to differing degrees. In the case of bacteriology, the specialisation is often governed by areas of application, such as medical bacteriology, plant pathogenic bacteriology, food applications, industrial use, and so on. Table 1.1 lists the major bacterial culture collections. Table 1.1. Major bacterial culture collections, listed alphabetically by continent and country

Collections AFRICA Rhizobium MIRCEN Department of Soil Sciences and Botany University of Nairobi PO Box 30197 Nairobi Kenya Holding: Rhizobia inoculants Rhizobium MIRCEN Centre National de Recherches Agronomiques Institut Senegalais de Recherches Agricoles BP51 Bambey Senegal Holding: Rhizobia AMERICA Fundacao Tropical de Pesquisas e Tecnologia 'Andre Tosello' Rua Latino Coelho, 1301 Caixa Postal 1889 13.100 Campinas, SP Brazil Telephone: (0192) 42-7022 Electronic mail: BT TYMNET 42:CDT0094 Holding: Industrial and general bacteriology Colecao de Culturas Adolfo Lutz Instituto Adolfo Lutz Av. Dr. Arnaldo, 355 CEP 7027 Sao Paulo Brazil Telephone: (011) 853-0111 Holding: General microbiology

Acronyms and World Data Center number (see Chapter 2)

MAO WDC-53

FTPT

IAL WDC-282

Resource centres Table 1.1. (cont.)

Collections AMERICA (cont.) Secao de Bacterias Fitopatogenicas Institute) Biologico Rodovia Heitor Penteado, km 4 Caixa Postal 70 13.100 Campinas, SP Brazil Telephone: (0192) 52-1657 Holding: Plant pathogenic bacteria

Acronyms and World Data Center number (see Chapter 2)

IBSBF

Institute) Nacional de Controle de Qualidade em Saude Fundacao Oswaldo Cruz Av. Brasil 4365 - Manguinhos Caixa Postal 926 20.000 Rio de Janeiro, RJ Brazil Telephone: (021) 270-1522 and 270-1072 Holding: Medical bacteriology

INCQS

Secao de Leite e Derivados Instituto de Tecnologia de Alimentos Av. Brazil 2880 13.100 Campinas, SP Brazil Telephone: (0192) 41-5222 Holding: Non-pathogenic bacteria

ITALSL

Secao de Microbiologia Instituto de Tecnologia de Alimentos Av. Brazil 2880 13.1000 Campinas, SP Brazil Telephone: (0192) 41-5222 Holding: Non-pathogenic bacteria

ITALSM

Instituto Zimotecnico Departamento de Tecnologia Rural/ESALQ Av. Padua Dias 11 Caixa Postal 9 13.400 Piracicaba, SP Brazil Telephone: (0194)33-0011 Holding: Non-pathogenic bacteria

IZ

6

L. R. Hill, K. Komagata and R. L. Gherna

Table 1.1. (cont.)

Collections AMERICA (cont.) Laboratorio de Fisiologia Bacteriana Fundacao Oswaldo Cruz Av. Brasil 4365 Caixa Postal 926 20.000 Rio de Janeiro, RJ Brazil Telephone: (021) 270-6565 Holding: Bacillus spp. Equipe de Microbiologia do Solo Instituto de Pesquisas Agronomicas (IPAGRO) Rua Goncalves Dias, 570 90.000 Porto Alegre, RS Brazil Telephone: (0512) 33-5411 Holding: Rhizobia Departamento de Antibioticos Universidade Federal de Pernambuco Cidade Universitaria 50.00 Recife, PE Brazil Telephone: (081) 271-3628 Holding: Non-pathogenic bacteria Instituto de Microbiologia Departamento de Microbiologia Medica Universidade Federal do Rio de Janeiro Av. Brigadeiro Trampowski, s/n Ilha do Fundao 20.000 Rio de Janeiro, RJ Brazil Telephone: (021) 260-4193 Holding: Medical bacteriology Salmonella Genetic Stock Centre Department of Biology University of Calgary Calgary Alberta T2N 1N4 Canada Holding: Salmonella spp. CIAT Rhizobium Collection Centro International de Agricultura Tropical AA 67-13 Cali Colombia Holding: Rhizobia

Acronyms and World Data Center number (see Chapter 2) LFB-FIOCRUZ

SEMIA WDC-443

UFPEDA

UFRJIM

LSCC WDC-338

CIAT WDC-536

Resource centres Table 1.1. (cont.)

Collections AMERICA (cont.) E. coli Genetic Stock Center Department of Human Genetics Yale University School of Medicine PO Box 3333 333 Cedar Street New Haven Connecticut 06510 USA Telephone: (203) 785-2687 Holding: E. coli, particularly K-12 derivatives, Hfr and F' strains Agricultural Research Service Culture Collection Northern Regional Research Center Agricultural Research Service, US Department of Agriculture 1815 North University Street Peoria Illinois 61604 USA Telephone: (309) 685-4011 Electronic mail: BT TYMNET 42:CDT0404 Holding: Mainly Streptomyces, Bacillus spp. IDA status American Type Culture Collection 12301 Parklawn Drive Rockville Maryland 20852 USA Telephone: (301) 881-2600 Telex: 898055 ATCC NORTH Electronic mail: BT TYMNET 42:CDT0109 Holding: General microbiology IDA Status Bacillus Genetic Stock Center Department of Biochemistry Ohio State University 484 West 12th Avenue Columbus Ohio 43210 USA Telephone: (614) 292-5550 Holding: Genetic strains of Bacillus spp., wild types and mutants

Acronyms and World Data Center number (see Chapter 2)

NRRL WDC-97

ATCC WDC-1

BGSC

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L. R. Hill, K. Komagata and R. L. Gherna

Table 1.1. (cont.)

Collections AMERICA (cont.) Neisseria Reference Laboratory Division of Infectious Diseases United States Public Health Hospital 1131 14th Ave S. Seattle Washington 98114 USA Holding: Neisseria spp.

Acronyms and World Data Center number (see Chapter 2) NRL

NIFTAL Rhizobium Germplasm Resource NIFTAL Project PO Box 0 Paia Hawaii 96779 USA Holding: Rhizobia

TAL WDC-506

USDA Rhizobium Culture Collection United States Denartment of Agriculture Beltsville Agricultural Research Center Beltsville Maryland 20705 USA Holding: Rhizobia

BRCC WDC-540

In Vitro International Inc. 611(P) Hammonds Ferry Road Linthicum Maryland 21090 USA Holding: General microbiology IDA status ASIA AND OCEANIA China Committee for Culture Collection of Microorganisms Chinese Academy of Sciences Institute of Microbiology Zhongguancun Beijing 100080 People's Republic of China Holding: General microbiology

IVI

CCCCM WDC-550

Resource centres Table 1.1. (cont.)

Collections ASIA AND OCEANIA (cont.) Food Industry Research and Development Institute PO Box 246 Hsinchu 300 Taiwan Telephone: (035) 223191-6 Holding: General bacteriology Persian Type Culture Collection Iranian Research Organisation for Science and Technology 118 Felestine Ave., Opp Kalantary 7 Tehran Iran Telephone: (021) 666777 Telex: TPBA 212918 Holding: General bacteriology Agency of Industrial Science and Technology Ministry of International Trade and Industry 1-3, Higashi 1-chome Tsukaba-gun Ibaraki-ken 305 Japan Telephone: (0298)54-6029 Telex: 3652570 AIST J Holding: General microbiology (excluding human pathogens) IDA status Culture Collection of the Institute for Fermentation Osaka Institute for Fermentation Osaka 17-85 Juso-Honmachi 2-chome Yodogawa-ku Osaka Japan Holding: General microbiology Kansai Medical School Culture Collection Kansai Medical School, Department of Microbiology Fumizono-Cho Osaka Japan Holding: General bacteriology

Acronyms and World Data Center number (see Chapter 2) FIRDI

PTCC

FRI

IFO WDC-191

KMS WDC-305

10

L. R. Hill, K. Komagata and R. L. Gherna

Table 1.1. (cont.)

Collections ASIA AND OCEANIA (cont.) Japan Collection of Microorganisms RIKEN Wako Saitama 351-01 Japan Telephone: (484) 62-1111 Telex: 2962818 RIKEN J Cable: RIKAGAKUINST Electronic mail: BT TYMNET 42:CDT0007 Holding: General microbiology Institute of Applied Microbiology University of Tokvo Yayoi 1-1-1, Bunkyo-ku Tokyo 113 Japan Telephone: 3812-2111 Holding: General and applied microbiology Philippine Type Culture Collection Bureau of Research and Laboratories Department of Health PO Box 911 Manila Philippines Holding: Medical bacteriology TISTR Culture Collection Thailand Institute of Scientific and Technological Research 196 Phahonyothin Road Bangkhen Bangkok 9 Thailand Telephone: 5791121-30 Telex:21392 TISTR TH Electronic mail: BT TYMNET 75:DBI0275 Holding: General microbiology AUSTRALASIA Department of Microbiology University of Queensland St Lucia Queensland 4067 Australia Telephone: (010617) 377-1111 Telex: 40315 UNIVQLD AA Electronic mail: BT TYMNET 42:CDT0113 Holding: General microbiology

Acronyms and World Data Center number (see Chapter 2) JCM WDC-567

IAM

WDC-190

PTCC WDC-46

TISTR WDC-383

UQM WDC-13

Resource centres Table 1.1. (cont.)

Collections AUSTRALASIA (cont.) New Zealand Reference Culture Collection, Medical Section National Health Institute Kenepuru Drive PO Box 50-348 Porirua New Zealand Telephone: (04) 370-149 Fax: (04) 378-983 Holding: Medical bacteriology; mycoplasmas, leptospiras International Collection of Micro-organisms from Plants Plant Diseases Divsion DSIR Mt Albert Road Private Bag Auckland New Zealand Holding: Plant pathogenic bacteria and fungi, agricultural microbiology EUROPE Bacterial Culture Collection Institute Tropical Medicine Nationalestraat 155 B-2000 Antwerpen Belgium Holding: Medical bacteriology Laboratorium voor Microbiologie en Microbiele Genetica Faculteit der Wetenschappen Ledeganckstraat 35 B-9000 Gent Belgium Telephone: (091) 227821 Telex: 12754 Electronic mail: TELECOM GOLD 75:DBI0242 Holding: General bacteriology, plant pathogens Bulgarian Type Culture Collection Institute for State Control of Drugs Zladimir Zaimov No. 26 1040 Sofia Bulgaria Holding: General microbiology, plasmids

Acronyms and World Data Center number (see Chapter 2) NHI

WDC-457

ICMP WDC-52

MCITM WDC-400

LMG WDC-296

BTCC WDC-66

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L. R. Hill, K. Komagata and R. L. Gherna

Table 1.1. (cont.)

Collections

Acronyms and World Data Center number (see Chapter 2)

EUROPE (cont.)

National Bank for Industrial Microorganisms and Cell Cultures Boul. Lenin 125BL2 Sofia III 3 Bulgaria Holding: Non-pathogenic bacteria; plasmids

NBIMCC WDC-135

IDA status

Collection of Animal Pathogenic Microorganisms Veterinary Research Institute Hudcova 70 62 123 Brno 21 Czechoslovakia Telephone: (05) 741121 Telex: 62475 Holding: Veterinary bacteriology, virology; mycoplasmas Czechoslovak Collection of Microorganisms Masaryk University Jostova 10 66 243 Brno Czechoslovakia Telephone: (05) 23407 Telex: 61241 VDS C Electronic mail: TELECOM GOLD 75:DBI0154 Holding: General microbiology Czechoslovak National Collection of Type Cultures Institute of Hygiene and Epidemiology Srobarova 48 100 42 Prague 10 Czechoslovakia Holding: Medical, veterinary bacteriology VTT Collection of Industrial Microorganisms VTT Biotechnical Laboratory Tietotie 2 SG-02150 Espoo Finland Telephone: (0) 4561 Telex: 12292 Holding: Non-pathogenic bacteria

CAPM WDC-181

CCM WDC-65

IEM WDC-130

VTT

Resource centres

13

Table 1.1. (cont.)

Collections EUROPE (cont.) Collection Nationale de Cultures de Microorganisms Institut Pasteur 25-28 Rue du Dr Roux F-75724 Paris Cedex 15 France Telephone: (1) 45-68-82-51 Telex: 250609 PASTEUR F Fax: (1) 43-06-98-35 Holding: Medical, veterinary bacteriology IDA status IMET Kulturensammlung Zentralinstitut fur Mikrobioloeie und Experimentelle Therapie Beutenbergstrasse 11 Jena 69 German Democratic Republic Telephone: (078) 852433 Telex: 5886142 Holding: General and medical bacteriology;

Acronyms and World Data Center number (see Chapter 2) CNCM WDC-174

IMET WDC-217

Nocardia spp.

Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg lb D-3300 Braunschweig Federal Republic of Germany Telephone: (0531) 6187) Fax: (0531) 618718 Electronic mail: TELECOM GOLD 75:DBI0225 Holding: General bacteriology IDA status Hungarian National Collection of Medical Bacteria National Institute of Hygiene Gyali Ut 2-6 H-1097 Budapest Hungary Telephone: (1) 142250 Holding: Medical bacteriology

DSM WDC-274

HNCMB WDC-258

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L. R. Hill, K. Komagata and R. L. Gherna

Table 1.1. (cont.)

Collections

Acronyms and World Data Center number (see Chapter 2)

EUROPE (cont.) National Collection of Agricultural and Industrial Microorganisms University of Horticulture and Food Industry Department of Microbiology Somloi Ut 14-16 H-1118 Budapest Hungary Telephone: (1) 665411 Electronic mail: TELECOM GOLD 75:DBI0185 Holding: Non-pathogenic bacteria IDA status

NCAIM WDC-485

Instituto do Patologia Vegetale (IPV) Culture Collection Cattedra di Micologia Via Celoria 2 1-20133 Milano Italy Holding: Actinomycetes, Streptomyces spp. Centraalbureau voor Schimmelcultures Royal Netherlands Academy of Arts and Sciences Oosterstraat 1 PO Box 273 NL-3740 AG Baarn The Netherlands Telephone: (2154) 11841 Electronic mail: TELECOM GOLD 75:DBI0218 Holding: Mainly Streptomyces spp. (also fungi, yeasts) IDA status WHO/FAO Collaborating Centre for Reference and Research in Leptospirosis Royal Tropical Institute Department of Tropical Hygiene Mauritskade 57 NL-0192 AD Amsterdam The Netherlands

IPV WDC-175

Holding: Leptospira spp.

CBS WDC-133

ITH WDC-196

Resource centres

15

Table 1.1. (cont.)

Collections EUROPE (cont.) Phabagen Collection Department of Molecular Cell Biology Section Microbiology State University of Utrecht Padualaan 8 Utrecht The Netherlands Holding: Genetic strains, plasmids Polish Collection of Microorganisms Polish Academy of Sciences Ul. Czerska 12 53-114 Wroclaw Poland Holding: Medical bacteriology Coleccion Espanola de Cultivos Tipo Departamento de Microbiologia Facultad de Ciencias Biologicas Universidad de Valencia Burjasot Valencia Spain Telephone: (096) 3630011 Fax: (96)3864372 Electronic mail: BT TYMNET 42:CDT0428 Holding: General microbiology Culture Collection of University of Goteborg Department of Clinical Bacteriology Guldhetsgatan 10 S-41346 Goteborg Sweden Telephone: (031) 602016 Electronic mail: TELECOM GOLD 75:DBI0070 Holding: Medical bacteriology Centre de Collection de Types Microbiens Rue du Bugnon 44 CH-1011 Lausanne Switzerland Telephone: (021) 223391 Holding: Medical bacteriology

Acronyms and World Data Center number (see Chapter 2)

PCM WDC-106

CECT WDC-412

CCUG

CHUV WDC-475

16 Table 1.1.

L. R. Hill, K. Komagata and R. L. Gherna (cont.)

Collections EUROPE (cont.) Culture Collection of Department of Microbiology Medical Faculty of Istanbul University Temel Bilimler Binasi, Capa-Topkapi Istanbul Turkey Telephone: (1) 5255504 Holding: Medical bacteriology National Collection of Food Bacteria National Institute for Research in Dairying University of Reading Shinfield Reading Berkshire RG2 9AT UK Telephone: (0734) 883103 Telex: 265871 Electronic mail: TELECOM GOLD 75:DBI0013 Holding: Agricultural, food, veterinary bacteriology National Collection of Industrial and Marine Bacteria Ltd 23 St Machar Drive Aberdeen AB2 1RY UK Telephone: (0224) 273332 Telex: 73458 UNIABN G Fax: (0224) 487658 Electronic mail: TELECOM GOLD 75:DBI0271 Holding: General bacteriology (excluding medical), marine bacteria; plasmids IDA status National Collection of Plant Pathogenic Bacteria Ministry of Agriculture, Fisheries and Food Hatching Green Harpenden Hertfordshire AL4 2BD UK Telephone: (058) 275241 Telex: 826363 Fax: (058) 272254 Holding: Plant pathogenic bacteria

Acronyms and World Data Center number (see Chapter 2) KUKENS WDC-101

NCFB WDC-118

NCMB WDC-238

NCPPB WDC-126

Resource centres

17

Table 1.1. (cont.)

Collections EUROPE (cont.) National Collection of Type Cultures Central Public Health Laboratory Service 61 Colindale Avenue London NW9 5HT UK Telephone: (01) 200-4400 Telex: 8953942 DEFEND G Fax: (081) 200-7874 Electronic mail: TELECOM GOLD 75:DBI0086 Holding: Medical, veterinary bacteria; plasmids; mycoplasmas IDA status Rothamsted Rhizobium Collection Welsh Plant Breeding Station Plas Goggerddan Aberystwyth SY23 3EB Dyfed UK Holding: Rhizobia The Wellcome Bacterial Collection Wellcome Foundation Limited Langley Court Beckenham Kent BR3 3BS UK Telephone: (081) 658-9966 Telex: 23937 WELLAB Electronic mail: TELECOM GOLD 75:DBI0510 Holding: Medical, veterinary bacteriology USSR Collection of Microorganisms Research Institute for Genetics and Industrial Microorganisms Breeding Doroshnaya Street, No. 8 113545 Moscow USSR Holding: Non-pathogenic bacteria IDA status

Acronyms and World Data Center number (see Chapter 2) NCTC WDC-154

RCR WDC-163

WRL WDC-142

VKM WDC-342

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L. R. Hill, K. Komagata and R. L. Gherna

Table 1.1. (cont.)

Collections

Acronyms and World Data Center number (see Chapter 2)

EUROPE (cont.)

USSR Collection of Microorganisms Institute of Biochemistry and Physiology of Microorganisms Academy of Sciences Pushchino-na-Oke 142292 Moscow Region USSR Telephone: (095)2316576 Fax: (095)9233602 Holding: Non-pathogenic bacteria

VKM

WDC-342

IDA status

USSR Collection of Microorganisms Research Institute of Antibiotics Nagatinskaya Street 3-a 113105 Moscow USSR Holding: Medical bacteriology (but excluding human and animal pathogens)

RIA

SDC-337

IDA status

The service collections usually levy fees for the supply of cultures, partially off-setting costs but not to the detriment of providing a service to the scientific community. A balance has to be struck between levying fees at a level which is economic in a commercial sense and that reminds the user that a purposefully conserved culture involves real expenditure, and a 'workable' level, given the nature of the resource. Viable organisms are re-usable and can be subcultured, which may lead users to obtain 'authentic' cultures at second or third hand, jeopardising the very authenticity and purity of the cultures. Frequently, the service collections have fee structures which make a distinction between commercial and non-commercial users. The distinguishing feature of service collections resides, however, in having staff whose primary task is curatorial. Through continuity of work, these resource centres become expert in documentation and preservation techniques and in knowledge of the behavioural proper-

Resource centres

19

ties of their holdings. As most bacteria can be satisfactorily preserved by freeze-drying (lyophilisation: see Chapter 4), the preparation of stocks for off-the-shelf supply is exploited so as to make time available for other necessary curatorial duties. For a service collection to be a truly valuable resource centre, it must keep abreast of the developing science it services. There must be an active accessioning policy, ongoing re-assessment of preservation procedures, awareness of taxonomic changes, identification of new requirements. Most of the service collections listed in Table 1.1 are able also to carry out original scientific research; this is important in itself, but also in attracting and keeping motivated staff without whom a service collection risks becoming fossilised and inevitably declines in utility as a resource centre. 1.3.2

Specialist collections

There are a great variety of specialist collections of bacteria. Some may be of a size, in terms of numbers of cultures maintained, comparable to some of the service collections. If their purpose is purely private, for example the in-house collection of an industrial company, then they are not strictly resource centres for the wider scientific community, although the conservation and documentation secured in these make them potential resource centres. Many specialist collections are the result of research programmes, probably comprising multiple exemplars of a limited number of species. In medical bacteriology there are instances where epidemiological typing of isolates calls for infra-subspecific (e.g. serotype, biotype, phage-type) identifications and a specialist collection may consist of just one species, such as a set of Staphylococcus aureus bacteriophage propagating strains and the corresponding bacteriophages. The specialist research collections also need to be documented to be valuable resource centres. They may not have an associated permanency if they are neither a large in-house collection nor designed for a particular application; upon cessation of the research activity that originated a collection, it may languish or become lost. It is not always practicable, nor even desirable, for a service collection to assume responsibility for every endangered collection. However, through special committees set up by the national and international organisations of culture collections (see Chapter 8), there is a growing awareness of the importance of specialist collections and a recognition of the need to safeguard them against loss. It is here that adequate documentation becomes of paramount importance in order to distinguish between

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L. R. Hill, K. Komagata and R. L. Gherna

resources that are unique and those that are commonplace or already adequately represented in other collections, in whole or in part. The community of culture collections is becoming even more closely knit through federations, both nationally, regionally, and world-wide and the secretariats of these provide access points for first enquiries (see Chapters 2 and 8). Telephone contact with service collections is generally restricted to simple queries. Where time permits, complex enquiries - especially if related to properties of cultures or suitability for an innovative application - are best done by correspondence. This allows the service collection to check more thoroughly the information at hand, or locate where else an answer can be obtained. An authoritative answer may involve cross-checking with many catalogues, looking up strain data records or contacting known experts. 1.4

Future development of resource centres One of the benefits of the current upsurge in biotechnological activity is an increased awareness by administrators that culture collections are an integral part of the infrastructure of microbiology. As resource centres, they always have had this role; but the speed at which biotechnology is now developing draws attention to the concurrent need to upgrade collections. Most of the service collections are publicly funded and their financial needs to meet the new demands must therefore compete with other publicly funded activities. That there is a solid pre-existing base on which to build augurs well. Certain developments, for example utilisation of computers in a variety of ways, are almost inevitable, but others are less obvious. A service collection, with its own in-house accessibility to a wide selection of cultures, is ideally placed to carry out contract work for third parties, and the development or evaluation of miniaturised identification kits is a single example. In order to satisfy the requirements of a more biotechnologically orientated user clientele collections need to acquire skills in molecular biology techniques, and several are developing in this direction. The need to accession genetically manipulated strains, vectors, or plasmidbearing strains is paralleled by a need by the collection to introduce appropriate quality control procedures additional to those already in existence as standard practice. The appointment of additional specialised staff on fixed-term contracts, is beneficial in two ways. First, it allows the collection to augment its research activity and acquire additional skills; but second, new staff members become familiar with the

Resource centres

21

full range of collection activities, gain a fuller knowledge of the significance of resource centres, and become trained in preservation skills and appreciative of the needs for documentation, all of which will be beneficial to their future employers. Permanent staff of service collections frequently serve as teaching faculty on training courses and it is probable that another future development of service collections will be an increased commitment to training. The need for technology transfer of culture collection expertise is increasingly recognised. 1.5

Reference Staines, J. E., McGowan, V. F. and Skerman, V. B. D. (eds.) (1986). World Directory of Collections of Cultures of Microorganisms, 3rd edition.

University of Queensland: World Data Center.

Information resources M. I. KRICHEVSKY and H. SUGAWARA

2.1

Introduction Microbiologists are faced with consideration of exponential growth in their laboratories on a daily basis. As users of a chapter on information resources for biotechnology they are exposed to a double dose of exponential growth. First, the explosion of information technology itself is due to the massive amounts of computing power available at ever diminishing cost. In turn, a population of computer aware and computer literate microbiologists represent a growing demand for more sophisticated access to modern information technology. The community of information technologists in concert with microbiologists are responding to this demand with a multiplicity of initiatives using various strategies. The resulting activity induces feelings of inadequacy in the authors of such chapters as this, since at the moment of delivery to the editors the information is out of date. Resources previously known only by rumour are tested. Simple facilities being tested as pilot projects are quickly made available to the community. Local data banks open their doors to regional and even world-wide participation. Databases on databases spring up because of the need to discover available resources. The net result is an ever increasing base of information resources for biotechnologists. In some cases, useful resources fall by the wayside, as have at least two of the resources listed. They have been discontinued in the interval between the first and present versions of this chapter. The root cause of such discontinuing of effort is lack of appreciation by the initial funding bodies of the complexity and time scale involved in database initiatives of this sort. 22

Information resources

23

While the information about information presented in this chapter is out of date as soon as it is written, the resources described are most likely to be improved and be more useful than the descriptions indicate. For information on new developments, the listed resources should be contacted. 2.2

Information needs The need of the biotechnologist for widely disparate categories of information is a consequence of the varied nature of the tasks required to design, develop, and consummate a process. The biotechnologist must find or develop genotypes of the required composition, discover the conditions for expression of the desired phenotypic properties, maintain the clones in a stable form, and describe all of these parameters in a fashion understandable to peers. Most of the categories of information required will be outside the expertise of any one individual. Thus, a panel of experts representing all disciplines involved must be assembled or access to diverse databases must be achieved. The library or publicly accessible databases can lead to the required information sources which range from the traditional scholarly publications to assemblages of factual or primary laboratory observations. This chapter presents an overview of the kinds of information available and the mechanisms to access them. In particular, it will concentrate on information resources for finding material with the desired attributes, be they taxonomic, historical, genetic, or phenotypic. 2.2.1

Interdisciplinary information sources

The personal training and professional experience in the particular narrow field in which they work allows microbiologists to perform many daily tasks without reference to outside sources of information. However, the interdisciplinary nature of the practice of biotechnology forces the use of navigational aids to the knowledge base of unfamiliar areas. This point is demonstrated by the observation that approximately 80% of the enquiries to the CODATA/IUIS Hybridoma Data Bank (see below) are from persons who are not immunologists. It follows that a successful database resource should be designed with the interdisciplinary users in mind as they often will form the largest segment of the user population. The main pathways to locating an existing source of strains with the properties that will be useful in the projected process are through

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records of the primary observations of properties or through derived information such as taxa or strain designations. In either case, the desired result is one or more strain designations and instructions on where to get cultures. Even though the desired end result is the same with both pathways, the mechanisms for recording and disseminating the information are usually, but not necessarily, quite different. Culture collections which have a stated mission of providing service to a public user community, especially through distribution of cultures outside their host institution, tend to use a taxonomic orientation in that their records are commonly kept as discrete strain descriptions, often one strain to a page. The whole strain description is easily read while comparisons among strains are difficult. Culture collections serving predominantly as local institutional repositories of strains for research, teaching, or voucher specimens for archival storage, tend to rely on primary observation data kept in tabular form with the attribute designations as column headings and the strain designations as row labels. In contrast to the previous case, comparisons among strains are easily made, while assembling a complete strain description may require following the row designation for the strain through multiple tables. Even now, most service collections use traditional paper-based data management methods rather than computers. Within a few years, the majority of collections will be using computers as their main datahandling tool. The functional distinctions between these alternative forms of data organisation blur with the use of computers, but can still be a factor if good information management practices are not followed. 2.2.2

Where to get strains

The ultimate source for strains with desired properties is isolation from nature. Indeed, large efforts have been mounted to find strains with desired properties such as production of antibiotics. These efforts are feasible when a good screening procedure, such as zones of clearing around a colony, is available. Even then, the effort is labourintensive with resulting high costs. The existence of culture collections with data on the characteristics of the holdings makes possible enormous savings when appropriate strains are available. However, the data must be available to the process developer with reasonable ease. The best source for strains will often be from the collection with the most available data rather than the most complete selection of strains with likely sets of characteristics.

Information resources

25

2.2.3

How to get strains The pathways used in obtaining access to the required data for finding desirable strains start with the same foci as the collections themselves. A taxonomic strategy or primary observation strategy may be used. A taxonomic search strategy for strains producing higher concentrations of a particular material (e.g. penicillin, riboflavin, ethanol) might well begin with asking service culture collections for all strains of Penicillium notatum, Ashbya gossypii, Saccharomyces cerevisiae in their collections and screening them for level of production. This method of searching requires the searcher to know which taxa are likely to have the desired attributes. A primary observation search strategy for organisms with the ability to degrade a particular material might well start with asking for all strains that degraded that material and had the growth characteristics that were desirable under the projected process conditions. The question to the collection might be 'Could you provide me with the characteristics of all your strains able to use hexadecane while growing aerobically at 25°C?' This method of searching requires no taxonomic knowledge on the part of the searcher. Clearly, the format of data storage in the collection would markedly affect the relative ease of answering these questions. 2.2.4

Strain data The traditional classes of data used to describe strains in culture collections still predominate. These include morphological, physiological, biochemical, genetic, and historical data. Clearly, these classes will form the basis for all future collection data as well. The current emphasis on biotechnology places new demands on the informational spectrum desired of microbial and cell line collections. In addition to the requests for taxa with specific properties, information is also requested on potential utility of strains in biotechnological processes and the actual or potential hazards arising from their use. These ancillary but important data are sparse in their availability, but some service collections with a concern for the needs of industry are collecting data of attributes specifically useful in biotechnological processes such as temperature tolerances, behaviour in fermenters, stability of monoclonal antibodies under adverse conditions, toxicity of products, and pathogenicity for unusual hosts. Physiology. The bulk of information included in publicly accessible

26

M. I. Krichevsky and H. Sugawara

databases will be physiological and biochemical. Most queries will be on what the strains may be capable of doing with respect to the desired process, and the databases will be heavily weighted towards containing this kind of information. While printed compendia of physiological and biochemical data are technically possible, they are rare and, in view of the expense of publishing such volumes, will continue to be rare. Rather, these kinds of data are more reasonably compiled in bulk and disseminated through computers. Morphology. The detailed morphological data held by most culture collections are generally of less interest than details of physiological data for process development. In various broad classes of organisms (fungi, protozoa, and algae) morphology may be critically important for taxonomy and identification. These same detailed attributes usually have little bearing on the conduct of most processes. However, some basic morphological information will be of fundamental importance in process engineering. For example, cell size knowledge is needed if filtration is a part of the process. Likewise, use of filamentous strains will require more energy for stirring fermenters than non-filamentous forms. Additionally, knowledge of sexual reproduction or fusion attributes is critical if hybridization or strain improvement programmes are to be carried out. Publicly accessible electronic databases used in searches will, with few exceptions, have only basic or general morphological information on strains because of the costs associated with printing or storage of such information. The decision on how much morphological detail to include in a public database will vary with the interaction between cost and importance of the information. Industrial. Data on the use of particular strains for specific processes is a frequently requested category of data. Such data exist, but in a widely scattered, uncoordinated fashion. They are contained in the open literature, collection catalogues, patent disclosures, and other more obscure repositories. The same is true of related data on strain behaviour in the processes themselves. There are few biological data compendia equivalent to the materials properties databases available to the engineers in chemistry, metallurgy, and similar disciplines. This situation is partly due to the nature of biological material and partly due to the development of high technology in biology later than in the other disciplines, in spite of the ancient history of biotechnology.

Information resources

27

Hazard. Risk assessment of biotechnological activities concerns governmental regulatory bodies throughout the world. All of the concerns are environmental and may be in such forms as a specific disease of humans, animals, or plants, an undesirable imbalance in the environment, or production of an undesirable product. The data available to answer queries in the area of risk assessment are quite sparse. The most common category of useful data is the pathogenicity of strains. This is largely a strain-specific phenomenon, the degree of virulence varying from strain to strain, especially on serial propagation. Less commonly available are data describing strain persistence in various environments and toxicity of products and, rarest of all, perhaps, data to predict the effect of the introduction of strains into new environments. 2.2.5

Taxonomic data

The traditional and important method of constructing databases in service culture collections is with taxonomic orientation. The storage of the data and their public presentation considers the strains first as representatives of their taxa. Further, the data given in the description of each strain in the catalogue of collection holdings are sparse as they depend on the assumption that the reader knows, or is adept at finding, the usual attributes of the taxon. Many clinical microbiology laboratories only save the phenotypic data on antibiotic resistance patterns and the putative name of the isolates; the data used to decide the name are discarded. The records may indicate that the isolate is 'atypical' without any indication of the attributes that led to this description. Given the traditional organisation of culture collection catalogues, the most common questions asked of service culture collections are likely to be on specific attributes of strains listed in the catalogues, or the companion question on the availability of strains with specific attributes. Indeed, experience shows that service collection personnel quickly develop great skill in searching the collection database by any and all means available to answer such questions from the community. In turn, the community learns that the collections are a valuable source of various kinds of information beyond that contained in the catalogues. The taxonomic orientation is natural in managing diverse culture collections and determining the boundaries of interest for many speciality collections. Within a large collection, curator responsibilities are usually assigned along taxonomic lines.

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M. I. Krichevsky and H. Sugawara

Historically, many service collections were established for studying or supporting the study of taxonomy. This essential function still underlies a great deal of service collection activity. Many of the laws and regulations concerning shipment, hazard, standards, laboratory safety, patents, and use of biological material are stated all or in large part in taxonomic terms. The US Federal Register refers to strain and species in defining which strains are to be used as standards for antibiotic testing. The attributes of these strains are not listed. Similar regulatory documents exist for other parts of the world. 2.2.6

Regulations

The development of regulations for efficient and safe use of biotechnological processes to the common good is heavily dependent on adequate data of diverse types. Unfortunately, databases do not exist to allow detailed appraisal of the potential hazard on a case-bycase basis. The use of taxonomic levels to evaluate and regulate is contraindicated by the very nature of the process of establishing taxa. The only solution to this dilemma requires the gathering and dissemination of appropriate data. 2.3

Information resources

An informal infrastructure of information gatherers, managers, and disseminators exists to answer questions on the practice and regulation of biotechnology as it relates to microbial strains and cell lines. It forms a very useful resource in spite of its informality. Further, a number of initiatives are under way which aim to manage this support system for biotechnology in a more formal and complete fashion. The infrastructure exists independently of the practice of biotechnology since the same needs for information transfer are basic to all of microbiology and cell biology. Collection holdings are raw material for these sciences and new information initiatives are stemming from the application of molecular biology and advances in biotechnology. These developments are merely refining the information pathways that evolved before modern genetic engineering focused the public eye on one of the oldest forms of manufacturing, the use of biological materials in processes. The historical sequence of development of the infrastructure started with the culture collections, proceeded with catalogue production, followed by collections of strain data in computers and, most recently, the creation of national, regional, and finally international data services.

Information resources

29

The ultimate source of data needed by the biotechnologist is the laboratory records of the collections. All other elements of infrastructure function to make these data accessible. The entry points to the pathways at all levels are the same as those considered previously: taxonomic or by detailed pattern of attributes. A combination is possible. The question 'Do you have a pseudomonad that degrades hexadecane?' will eliminate all yeasts that have the same ability. 2.3.1

Culture collection catalogues Service collections publish catalogues describing their available strains to inform the public of their holdings and the salient properties of strains. A secondary effect is to reduce some of the labour overhead involved in answering questions from the public. A number of nations (such as Japan, China, and Brazil) have prepared combined catalogues, eliminating the need to obtain and consult multiple catalogues. By the very nature of printed catalogues they function imperfectly since the information describing each strain is limited. Only one or at best a few attributes can be indexed so that detailed searching is impossible. Because of the positive accessioning policies of the service collections, catalogues are out of date upon publication. In general, the taxonomic entry route is served well, but the attribute route is necessarily left to follow-up questions to the collection itself. The most important deficiency is that only a very small proportion of the world's collections publish catalogues at all, since provision of a public service is not their prime function. Where catalogues do exist, they form an important resource for the biotechnologist. The information they contain is carefully presented. If a taxon is known, much time can be saved by consulting a catalogue for availability. Often, valuable ancillary information on use, literature references, propagation conditions, or patents is included in the catalogue. Finally, the staff of the collection listed in the catalogue can be contacted for further information on their holdings or as entry into the rest of the informal information services of the collection community. 2.3.2

Individual collections Culture collections of importance in biotechnology are not limited to the recognised service collections. In many cases, the biotechnologist must have access to a detailed collection of strains with the final selection of the particular strain for use in the process decided by personal comparative testing. Service collections may serve in this respect, but because of their usually broad nature cannot always main-

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M. I. Krichevsky and H. Sugawara

tain the detailed holdings of the personal research or survey collection. The mechanics of obtaining information from primary records of individual collections may not be simple. The curator must scan tables or individual strain records to match the pattern of the query. The process is faster for those collections which keep their records in a computer. While computer-aided searching is faster, considerable time is required of collection personnel to search on a query by query basis. Time spent in searching the database to answer public queries may be considered a normal and reasonable function by the service collections or a burdensome chore by collections with other basic missions. Either way, such searches can represent a considerable overhead on staff time. 2.3.3

Strain data compendia The availability of computers for both record keeping and data analysis has resulted in compendia of strain data in locations other than the collections. Hospitals have contributed antibiotic resistance data to large-scale surveys of changes of resistance plasmid distributions in bacterial populations. Numerical taxonomists assemble large databases of strain data in the course of their activities, often containing data contributed by other research workers. Ecologists and public regulatory agencies conducting surveys of the environment, including habitats such as soils, waters, foodstuffs, and wild and cultivated plants, often amass considerable amounts of data which are installed in some computer resource for management and analysis. The result is that the data describing the strains reside in a different location from the strains. Arising from all this activity is a body of curators of data in support of the curators of strains. The relevant information specialists may be associated with a collection, and where such data management exists, the biotechnologist's search is immensely facilitated. Since the taxonomic designation is managed in the computer in the same way as any attribute of the strain, the entry into the database can be taxonomic or by attribute pattern with equal facility. The problem of finding the strains of interest is largely reduced to the problem of finding the databases themselves. 2.3.4

National, regional, and international data resources Scientists and technologists band together in organisations focused on their disciplines in order to exchange information. Many of

Information resources

31

these scientific and technical societies also become providers of services to their members and the public community. Some resemble guilds or unions in providing advocacy for improving conditions for their members. Recently, societies have come full circle in that they are providing, directly or through advocacy, informational resources in the form of publicly available databases. Since most societies are national, the earliest of these database efforts were national as well. In some disciplines, national efforts were deemed so valuable that they became international in use. The Chemical Abstracts Service of the American Chemical Society is a well known example. Geopolitical regions have their counterparts in scientific and technical activities. The most notable of these regional efforts is within the European Economic Community with a ripple effect to other countries in Europe. Either by combining regional resources or by direct international efforts, world-wide database resources are being established in many disciplines. Some stand alone and others are distributed in networks. Informational resources describing culture collections and their holdings are distributed through all three levels as are the organisations concerned with sponsoring such resources. National and regional organisations concerning culture collections. Micro-

biologists having interests in culture collections have banded together in national federations for culture collections. In some cases, cell biologists also are included. The boundaries for membership seem generally loose. The countries that have such national federations are listed and further described in Chapter 8. The countries are Australia, Brazil, Canada, China, Czechoslovakia, Japan, Korea, New Zealand, Turkey, United Kingdom, and United States of America. Japan, Brazil, and the UK are most actively pursuing national databases on collection holdings. Others are being discussed or planned. The Japanese database is designed to contain information from all types of microbial collections. The Brazilian and UK systems are initially designed to concentrate on service collections, but could be expanded to include other collections as resources become available. The European Community has recently established a regional information service; the east European countries are actively planning networks on national and regional lines. Other databases are being discussed or planned and more details of these systems are given below.

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M. I. Krichevsky and H. Sugawara

Brazil

The third edition of the Catalogo Nacional de Linhagens was produced by the Fundaga Tropical de Pesquisas e Tecnologia 'Andre Tossello' in Campinas, Brazil in 1989. The species names and designations of strains held by Brazilian collections are listed in the catalogue. The catalogue includes bacteria, filamentous fungi, yeasts, protozoa, algae, animal cell lines, viruses, and miscellaneous microorganisms. The on-line version of the Catalogue is available on the Base de Dados Tropical, which is installed on the Brazilian national information system, Embratel and is accessible through international telecommunication systems with the proper access codes and billing arrangements. In addition to catalogue information, the BDT contains a directory of specialists and research projects in applied microbiology in Brazil. Contact:

Funda^ao Tropical de Pesquisas e Tecnologia 'Andre Tosello' Rua Latino Coelho, 1301 13.100 Campinas, SP Brazil Telephone: (00192) 42-7022 Electronic mail: BT TYMNET 42:CDT0094 European Culture Collections' Organisation (ECCO)

ECCO is an active group comprised of representatives of major service collections in countries that have microbiological societies affiliated with the Federation of the European Microbiological Societies (FEMS). All the ECCO collections are also affiliated with the World Federation for Culture Collections. Forty-one collections represented are from Belgium, Bulgaria, Czechoslovakia, Finland, France, Federal Republic of Germany, German Democratic Republic, Greece, Hungary, Italy, Netherlands, Norway, Poland, Portugal, Spain, Sweden, Switzerland, Turkey, United Kingdom, Union of Soviet Socialist Republics and Yugoslavia. ECCO was established in 1982 to collaborate and trade ideas on all aspects of culture collection work. Since service collections inherently are organised repositories of information as well as cultures, ECCO is a valuable resource for finding information of interest in biotechnology. Each of the collections produces a catalogue of holdings. Such catalogues often hold information beyond the listing and description of the

Information resources

33

strains held. Also, the curators are likely contacts for other, non-service collections in their countries. As intercollection communication pathways grow and become formalised within and between ECCO countries, the prospects for an electronic communication network encompassing all these countries grows as well. Such a network is being actively discussed among its members at this time. ECCO members are collaborating with the Information Centre for European Culture Collections (see below). Contact:

USSR Collection of Microorganisms Institute of Physiology and Biochemistry of Microorganisms Academy of Sciences Pushchino-na-oke 142292 Moscow Region USSR Telephone: (095) 2316576 Fax: (095) 9233602 Japan Federation for Culture Collections (JFCC)

JFCC has promoted cooperation among culture collections and individuals in Japan as well as internationally since 1951. In 1953, the JFCC started collecting data on the holdings in Japan and completed the catalogue, which listed about 22 000 strains from 144 research institutions. The strains in the list were reidentified by a project team organised by Professor Kin'ichiro Sakaguchi of the University of Tokyo, resulting in the publication of a series of JFCC catalogues in 1962, 1966, 1968, and the most recent in 1987. The JFCC produces a bulletin that is published regularly. Contact:

JFCC NODAI Research Institute Culture Collection Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagaya-ku Tokyo 156 Japan Microbial Resource Centres (MIRCENs)

Information on Microbial Research Centres (MIRCENs) will be found in Chapter 8. These centres are found in both developed and develop-

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M. I. Krichevsky and H. Sugawara

ing countries. Each has its special focus of interest, for which it acts as a regional centre. National and regional data resources Information Centre for European Culture Collections (ICECC)

In 1988 the Commission of the European Communities provided funds under its Biotechnology Action Programme for the establishment of an information centre for European culture collections. An independent organisation, the centre is a useful source of information on all aspects of culture collection activity in Europe and beyond. Its mission is to hold information on holdings and specialised services, such as identification or patent services, available from the service collections in Europe. In addition it promotes the services of the collections at conferences and exhibitions, plans training courses and is able to act as a Secretariat for European culture collections. The MiCIS database (see below) was transferred to the ICECC in 1989 and is now available on-line. It is necessary to register with the ICECC to obtain access to the database. It is planned that this database will form the nucleus for a centralised European database holding data on individual strain properties. A gateway between MSDN and ICECC operates, giving users the opportunity to access the Centre either direct or through the MSDN network. Contact:

Information Centre for European Culture Collections Mascheroder Weg lb D-3300 Braunschweig Federal Republic of Germany Telephone: (0531) 618715 Fax: (0531) 618718 Electronic mail: TELECOM GOLD 75:DBI0274 Institute for Physical and Chemical Research (RIKEN)

In Japan, RIKEN carries out international activities, such as being the host institution for the World Data Center on Collections of Microorganisms and a node of the Hybridoma Data Bank. These activities are carried out by the Life Science Research Information Section (LSRIS) and Japan Collection of Microorganisms (JCM). On a national level, LSRIS has developed the National Information System of Laboratory Organisms (NISLO), a directory of Japanese collections and their holdings. The NISLO covers microorganisms,

Information resources

35

animals, and plants. In the case of microorganisms, the LSRIS closely cooperates with the JCM. The following information and services are currently available from LSRIS: (1) the number of laboratory animals used in Japan and their scientific names; (2) microorganisms maintained in the member collections of JFCC; (3) algae maintained in culture collections in the world; (4) identification of deciduous trees; (5) fundamental references for cell lines widely used in Japan; (6) bibliographical information for plant tissue and cell cultures. Contact: LSRIS RIKEN 2-1 Hirosawa, Wako Saitama 351-01 Japan Telephone: (0484) 621111 Electronic mail: BT TYMNET 42:CTD0007 Microbial Culture Information Service (MiCIS)

One of the first (along with Japan) national data efforts is that of the Microbial Culture Information Service (MiCIS) in the UK. This initiative is oriented towards providing public access to primary observation data while retaining the taxonomic orientation for computer entry of the data. The initiative is a cooperative effort between the UK Department of Trade and Industry (DTI) and the UK Federation for Culture Collections. The following description is quoted from a joint statement of purpose between MiCIS and MINE (see below). This database has been developed by the Laboratory of the Government Chemist (LGC) on behalf of DTI following consultation with industry. MiCIS initially contains all data currently available on strains including catalogue information, hazards, morphology, enzymes, culture, conditions, maintenance requirements, industrial properties, metabolites, sensitivities and tolerances. Further categories of information will be added as the system develops. MiCIS will contain data from all UK national culture collections and discussions are currently taking place to include data from private and other European collections.

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The funding for the MiCIS effort was terminated in 1989. The database is not being expanded or actively maintained by the culture collections at this time. However, the database has been transferred and is available on-line from the Information Centre for European Culture Collections (see above). It is also available on-line through the Microbial Strain Data Network (see below). Contact:

Information Centre for European Culture Collections Mascheroder Weg lb D-3300 Braunschweig Federal Republic of Germany Telephone: (0531) 618715 Fax: (0531) 618718 Electronic mail: TELECOM GOLD 75:DBI0274 Microbial Information Network Europe (MINE)

Within the European Economic Community (EEC) the regional activities are primarily focused within the programmes of the Commission of the European Community (CEC). They fund a number of initiatives, either as sole efforts or as collaborations when the initiative has a scope beyond the confines of the EEC. One such initiative within the EEC is MINE. MINE has the traditional taxonomic orientation of the service collections. The UK node has provided the following description of MINE in a joint (with MiCIS) statement of purpose: This EEC database is a computerised integrated catalogue of culture collection holdings in Europe and is prepared as a part of the EEC Biotechnology Action Programme. CMI [Commonwealth Mycological Institute], in collaboration with CABI [Commonwealth Agricultural Bureaux International] Systems Group, is to act as the UK node, in parallel with national nodes being developed in The Netherlands, Germany, and Belgium; discussions with Portugal and France are currently under way. . . . it will not include full strain data but only a minimum data set. Once enquirers locate a culture, they will then be referred to the collection concerned, or to national strain data centres . . . for any detailed strain information if this is needed. Initially, enquirers will contact MINE nodes by mail, telex,

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or telephone. At a later stage, on-line services may also be available on subscription. Some of the above statement may need to be modified with the passage of time. A hard copy integrated catalogue and minimum data set are deferred. Rather, MINE has become a cooperating group of collections. Various collections within MINE have established their own databases using similar software and agreed upon fields. Others have databases that are not directly compatible with others. Further, some of the collections have advanced to the point of having independent on-line systems available for public enquiry. There is agreement within MINE for support of a centralised database with full data set of strain characteristics, possibly within the framework of the Information Centre for European Culture Collections and based upon the MiCIS database now converted to MINE format and maintained at the Centre. Contact:

Information Centre for European Culture Collections Mascheroder Weg lb D-3300 Braunschweig Federal Republic of Germany Telephone: (0531) 618715 Fax: (0531) 618718 Electronic mail: TELECOM GOLD 75: DBI0274 Nordic Register of Microbiological Culture Collections

In 1984, the Nordic Council of Ministers initiated support for a Nordic Register of Culture Collections encompassing Denmark, Finland, Iceland, Norway, and Sweden. The Register's scope is inclusive of all sizes and functions of collections from small personal collections to large service collections. The first three years of development were focused on strains of importance to agriculture, forestry, and horticulture. The building of the microcomputer-based database was an undertaking of the Department of Microbiology of the University of Helsinki, Finland. Development of software was carried out in cooperation with the Nordic Gene Bank for Agricultural and Horticultural Plants in Alnarp, Sweden. In 1987, the funding from the Council of Ministers ceased. For this reason the Nordic Register is not functional.

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International

resources

The primary focus for international culture collection information resources is through the International Council of Scientific Unions (ICSU) with headquarters in Paris, France. Various components of ICSU have current or potential initiatives relating to providing information of interest to biotechnologists. These include: Committee on Data for Science and Technology (COD AT A), World Federation for Culture Collections (WFCC), International Union of Immunological Societies (IUIS), and International Union of Microbiological Societies (IUMS). Committee on Data for Science and Technology (CODATA)

CODATA was established in 1966 by the International Council of Scientific Unions (ICSU) to promote and encourage the production and international distribution of scientific and technological data. Its initial emphasis was in physics and chemistry, but its scope has been broadened to data from the geo- and biosciences. CODATA is 'especially concerned with data of interdisciplinary significance and with projects that promote international cooperation in the compilation and dissemination of scientific data'. The main activities of CODATA are carried out by Task Groups established for specific projects. Of special interest to biotechnologists are the biologically oriented Task Groups on the Hybridoma Data Bank, Microbial Strain Data Network, and Coordination of Protein Sequence Data Banks. The first two are the policy boards of the activities while the last is a coordinating body among the existing sequence data banks. The latest initiative of CODATA in biological information is the creation of a Commission on Terminology and Nomenclature in Biology. The purpose of the Commission is to locate existing standards of terminology or nomenclature in the disciplines of biology, publicise the existence of the standards, and encourage the development of standards where they are deemed lacking. The Commission has sought and obtained the cooperation of most of the constituent biologically oriented Unions of the International Council of Scientific Unions. While the development of standards is the responsibility of the Unions, the Commission will make the considerable experience of its members in developing computer-based vocabularies and databases in general available to the standard-setting bodies. The first such effort is in the area of viruses in collaboration with the International Committee on the Taxonomy of Viruses.

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Contact:

CODATA Secretariat 51 Boulevard de Montmorency F-75016 Paris France Telephone: (1) 45-25-04-96 Telex: 650553 Cable: ICSU PARIS Electronic mail: TELECOM GOLD 75:DBI0010 Hybridoma Data Bank (HDB)

In 1984, the CODATA/IUIS Hybridoma Data Bank (HDB) started building an international database on hybridomas, other immunoreactive cell lines, and monoclonal antibodies. The HDB is designed to act as a locator service, help avoid duplication of effort, and provide a research tool on relationships among reactivity patterns. An international infrastructure (with data bank branches in the USA, France, Japan, Canada, UK and India) is in place, a growing database is being assembled, and queries are being answered. A pilot project for a publicly accessible on-line service is in progress. Three publicly accessible on-line services are available: through the Microbial Strain Data Network (MSDN, see below) on BT TYMNET and TELECOM GOLD under the auspices of the American Type Culture Collection, in Canada through the National Research Council, and on DIMDI in the Federal Republic of Germany under the auspices of CERDIC. The most common queries involve the antigen-antibody reactivity/non-reactivity patterns. The possible antigens to be named cover all of biology, biochemistry, and a large part of organic chemistry. The complexities compound rapidly when such problems as tumour epitopes are at issue. The host taxonomy, organ, tissue, cell structure, developmental stage, pathology, antigen, and epitope all have nomenclatural ambiguities to some degree. Various authority sources, selected by exercise of best judgement, used in building a controlled vocabulary glossary reduce the problems to manageable proportions. Communication paths, conventions, and frequent quality control exercises facilitate consistency of response. The information on each cell line and product is quite comprehensive. In addition to reactivity attributes, considerable information is coded, such as fusion partners' histories, developer, availability, distributors), applications, assay procedures, antibody classes,

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immunisation techniques, literature and patent citations. Many of these categories have internal sub-categories. Queries to the HDB may be through mail, telephone, or through the on-line services listed above to any of the nodes listed below. More information on the HDB is available from any of the three nodes:

Hybridoma Data Bank 12301 Parklawn Drive Rockville, Maryland 20852 USA Telephone: (301) 231-5585 Electronic mail: BT TYMNET 42:CDT0004 LSRIS RIKEN 2-1 Hirosawa, Wako Saitama 351-01 Japan Telephone: (484) 621111 Telex: 2962818 RIKEN J Electronic mail: BT TYMNET 42:CDT0007 CERDIC Lab. d'Immunologie Fac. de Medecine Av. de Vallombruse F-06034 Nice France Telephone: 93-20-01-80 Electronic mail: TELECOM GOLD 75:DBI0014 Microbial Strain Data Network (MSDN)

The MSDN was started in 1985 in order to construct a world-wide network of holders of strain data serving as nodes in an informational network. The MSDN will act as a locator service for strains of microbes or cultured cell lines having specific attributes. From the UNEP-initiated round table in 1982 (at the International Congress for Microbiology), where the concept was introduced, to the present time, the Network has been designed. An infrastructure has been established, consisting of a policy-making Committee of Manage-

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ment, operating Committees, and a Secretariat; on-line databases have been installed on a publicly accessible commercial computer host. Sponsorship is by three components of the International Council of Scientific Unions: CODATA, WFCC, and IUMS. The sheer magnitude of the number of repositories of microbial strain data and the numbers of strains held within those repositories generate serious data acquisition and communication problems. Collecting the desired data in one or a few places for general availability is a practical impossibility. Rather, the MSDN is designed to operate as a locator service for repositories of strains with desired combinations of attributes by an indirect method. Thus, the data repositories become Network Informational Nodes. The Central Directory of the MSDN contains a list of the data elements recorded by all the various Nodes rather than the data themselves. This database is a controlled vocabulary of standardised nomenclature of mainly biochemical and morphological features used for strain characterisation throughout the world. The initial basis for the controlled vocabulary comes from the CODATA-sponsored publication by Rogosa, Krichevsky & Colwell (1986). The user scans the vocabulary to select features of interest. The features in the form of the controlled terms are entered as search criteria into a second database which yields the names of repositories assessing the strains for possession of the desired features. When a Node is located which codes information on the desired data elements (features), the person querying the MSDN contacts the Node(s) directly for existing strains fitting the detailed pattern of attributes. The contact may be accomplished through telecommunications (where the capabilities exist) or by mail or telephone. In an increasing number of cases, direct access to host computer databases is possible. In addition to the Central Directory, a number of other databases and services are available from the MSDN (see Tables 2.1 and 2.2). Thus, the HDB database provides a simple mechanism for locating hybridomas and/or monoclonal antibodies that their distributors state are available to the scientific community. Again, a number of culture collection catalogues are on-line with frequent updates. On-line ordering of cultures or requests for supplementary information on cultures is available from the culture collections maintaining a sales mailbox or catalogue on the system. Gateways to various collection computers have been established or are under development. The first one in place was to the National

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Table 2.1. MSDN services MSDN Central Directory Other databases (HDB, MiCIS, collection catalogues . . .) either stored on MSDN computer or made available through electronic gateway On-line culture ordering facility Electronic mail (including Telex and Fax) Bulletin board Computer conferences Micro-IS software distribution Training Access to BT TYMNET and TELECOM GOLD services (e.g. International Air Line Guide) Access to people on other BT TYMNET/TELECOM GOLD systems User support

Table 2.2. Databases available through the MSDN network

MSDN Central Directory - locates centres with specific information on the properties of microbial and cultured cells. A 'yellow pages' Directory referring users to sources of primary data. Data strictly defined in scientific terms, using a numeric coding system. World-wide data providers. Hybridoma Data Bank - contains data on publicly available immunoclones and their products (USA, Europe, Japan nodes) Information Centre for European Culture Collections (MiCIS and DSM databases)0 Databases of the Centraalbureau voor Schimmelcultures, Baarn, Netherlands* World Data Center on Collections of Microorganisms, Japan0 DATA-STAR databases0 ATCC Recombinant clones and libraries NCYC Computer Services0 Culture collection catalogues: American Type Culture Collection animal cells American Type Culture Collection algae and protozoa American Type Culture Collection bacteria CAB International Mycological Institute European Collection of Animal Cell Cultures UK National Collection of Food Bacteria (NCFB)0 UK National Collection of Yeasts (NCYC)0 0

Accessed via electronic gateway.

Collection of Yeast Cultures' computer. This gateway provides direct access to the catalogue as well as a yeast identification system. Another gateway is to the World Data Center for Collections of Microorganisms (WDC) in Japan, and the gateways to MiCIS, Deutsche Sammlung von

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Mikroorganismen databases, FRG and the Centraalbureau voor Schimmelcultures databases, is already operating. General gateways are available to the 'academic networks'. The electronic mail service on the MSDN system is a powerful communication tool in is own right. The System has integrated bulletin board and computer conferencing facilities. In addition the mail system is linked to the telex, cable and fax systems. Services of the MSDN that are not electronic include organising and conducting training courses in the use of computers in microbiology and international electronic communication, as well as distribution of the Microbial Information System (MICRO-IS) on a shareware basis. Queries to the MSDN may be through mail, telephone or through the BT TYMNET and TELECOM GOLD services which link to most common packet switching services as well as to telex and TWX systems. Contact:

MSDN Secretariat Institute of Biotechnology Cambridge University 307 Huntingdon Road Cambridge CB3 OJX UK Telephone: (0223) 276622 Telex: 81240 CAMSPL G Fax: (0223) 277605 Electronic mail: TELECOM GOLD 75:DBI0001/DBI0005 JANET: MSDN @ PHX.CAM.AC.UK World Data Center for Collections of Microorganisms (WDC)

In 1984, the World Federation for Culture Collections (WFCC) officially reaffirmed the WDC as a component of the WFCC and thus accepted responsibility for the operation and management of the WDC. Because of the announcement of the retirement of the director of the WDC at Brisbane, Australia, a public search was conducted for potential hosts to ensure continuity of the WDC effort. After competitive evaluation of the proposals, the Executive Board of the WFCC agreed that the WDC be transferred to the Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.

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M. I. Krichevsky and H. Sugawara

WDC is an information centre which supports culture collections and their users. The primary tasks are: (1) publication of descriptions of culture collections; (2) production of a species-oriented directory of culture collection holdings. The WDC publishing plans for 1989 included a World Directory of Algae (completed), a World Directory of Protozoa, and a Directory of Asian culture collections. The tasks of WDC are not limited to the above; other tasks will be performed based on necessity and available resources. Information sources for WDC are culture collections and national/local/international data centres as well. The WDC makes use of, and cooperates with, HDB and MSDN. For information dissemination, the WDC will use a variety of communication media such as mail, telex, cable, electronic mail, fax, publication and magnetic devices (floppy disks and magnetic tapes). The WDC currently holds information on 327 culture collections distributed over 56 countries. The core data of the WDC are descriptions of culture collections and their holdings, allowing users to locate a culture collection and/or taxonomic category of microorganism. They can find strains of specific species by consulting the list of species preserved in culture collections. The WDC database currently includes bacteria, fungi, yeasts, algae, protozoa, lichens, animal cells, and viruses. The WDC is able to answer queries by any communication media including on-line retrieval. Contact:

WDC/RIKEN 2-1 Hirosawa, Wako Saitama 351-01 Japan Telephone: (484) 621111 Telex: 2962818 RIKEN J Electronic mail: BT TYMNET 42:CDT0007 World Federation for Culture Collections (WFCC)

The World Federation for Culture Collections provides information to biotechnologists in a variety of ways within the overall mission of the WFCC as described in Chapter 8. Specific information initiatives des-

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cribed above are the World Data Center on Microorganisms and the Microbial Strain Data Network. Specialist committees established by the WFCC can provide information on a number of topics. The Patents Committee considers patent conventions. The Postal and Quarantine Committee is a good source of information on regulations governing shipment of cultures. The Publicity Committee publishes a newsletter on a periodic basis. The Committee on Endangered Collections identifies and takes action to rescue jeopardised collections. The Education Committee initiates training activities (e.g. books, videotapes, courses, individual instruction). For more information on the activities and information resources of the WFCC contact the WDC (see above), the MSDN (see above), or any of the resource centres listed in Chapter 1. Directory of Biotechnology Information Resources (DBIR)

There is a variety of newsletters, bulletin boards, and other information sources that cover various aspects of biotechnology in addition to those listed above. Finding these can be difficult in many instances. An initiative of the Specialized Information Services Division of the National Library of Medicine in the USA, in collaboration with the Bioinformatics Department of the American Type Culture Collection has the purpose of helping with this problem. The Directory of Biotechnology Information Resources (DBIR) is a centralised directory to international sources of publicly available biotechnology information such as: computerised databases and their distributors, networks, electronic bulletin boards, and other biological computer resources established for communicating and disseminating biotechnology data; culture collections and specimen banks; biotechnology centres and other organisations which stimulate biotechnology growth in academia and industry; publications focusing on general issues in biotechnology, which include selected directories, serials, monographs, reviews, and compilations; nomenclature committees working on issues of nomenclature in biotechnology and molecular biology. The Directory encompasses a broad spectrum of biotechnology, including its application in medicine, agriculture, pharmaceuticals, food technology and microbiology, as well as molecular biology. Because of the international and interdisciplinary scope of the Directory, it should prove especially useful to those requiring an entry point into unfamiliar areas of biotechnology.

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DBIR is currently maintained by MEDLARS on the National Library of Medicine's (NLM) Toxicology Data Network (TOXNET) and as a part of the Directory of Information Resources On-line (DIRLINE) file.

or

2.4 2.4.1

Contact: Directory of Biotechnology Information Resources Bioinformatics Department American Type Culture Collection 12301 Parklawn Drive Rockville, Maryland 20852-1776 USA Telephone: (301) 231-5585 Electronic mail: BT TYMNET 42:CDT0004 Specialized Information Services Division National Library of Medicine 8600 Rockville Pike Bethesda, Maryland 20894 USA Telephone: (301) 496-6531 or (301) 496-1131

Access to data resources Traditional Traditional methods for accessing data resources are still the most common and are quite satisfactory as long as the answers are not voluminous and are not needed quickly. Asking a culture collection curator about the availability of a single strain exemplifying a particular taxon or having a particular set of attributes will usually get a prompt reply. The query may be verbal, in person or on the telephone, or by mail. As the queries become more complex, the use of these methods becomes less satisfactory to both the seekers and providers of information. The seeker of information becomes frustrated at the delay and incomplete nature of the answer that comes back. Often multiple cycles are required to refine the question to the point where the desired answer is given. While this refinement of communication is valuable in clarifying the true nature of the query (not always recognised from the initial inquiry) and common to all pathways, the frustration is amplified by the length of the cycle time. The provider of information must devote increasing resources to answering queries. This takes professional expertise that could be used

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in the other work of the collection. Therefore, any mechanism which minimises the labour involved in answering queries increases the professional resources of the collection. Both internal and external mechanisms are useful to alleviate some of the workload. Internal mechanisms include publications, such as the aforementioned catalogues, and computer management of the data for ease of searching and reporting. External mechanisms are primarily electronic forms of communication and are discussed in the next section. 2.4.2

Electronic

Except for voice communication via telephone, telecommunication has only recently become a part of collection life. Many of the larger collections have been using printed electronic messages (cable, telex) for a while. However, the advent of computer-operated message transfer systems at reasonable cost (e.g. direct on-line access, electronic mail systems, and public packet switching services) have allowed economically practical electronic communication between questioner and answering resource. Two parallel paths of development are taking place at this time in providing public access to data in collections. Some collections are following both paths simultaneously. In the first instance, a collection may make its data accessible to the public by establishing access to a computer system maintained by the collection or its parent institution. Some examples are the Human Gene Probe Bank at the American Type Culture Collection, the National Collection of Yeast Cultures at Norwich, UK, the Japan Collection of Microorganisms, Tokyo, and the CAB International Mycological Institute, Kew, UK. The second approach is to install the data in a computer operated by others. The data and their installation may be accomplished under the control of the collection as is being done by the ATCC on the MSDN/CODATA Network. Alternatively, all or part of the data may be installed on a computer operated as a national or regional facility such as those described above for Europe, Brazil, and Japan (MiCIS, MINE, Catalogo National de Linhagens, and NISLO).

In all these cases of providing access through electronic computer services, the source of the data does not maintain the communication paths beyond the host computer. It is generally the responsibility of the seeker of information to find the most appropriate path. Unless the

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provider and seeker of information are at the same institution, where direct connection to the computer may be possible, the ordinary telephone system is likely to be the first resource connected to the seeker's terminal. Where short distances are involved, it may be reasonable to use only the voice-carrying telephone system. However, national and international data communications are using 'packet switching service' (PSS) for an ever increasing share of data telecommunications. Such PSS transmission is cheaper and more reliable by far than direct telephone calls. To find out more about telecommunications with the systems described in this chapter, get in touch with the appropriate system listed. Help with establishing electronic communication paths may be obtained from the MSDN (see above). 2.5

Reference Rogosa, M , Krichevsky, M. I. and Colwell, R. R. (1986). Coding of Microbiological Data for Computers. New York: Springer-Verlag.

3 Administration and safety L. R. HILL and R. L. GHERNA

3.1 3.1.1

Administration Supply of cultures The aim of service culture collections is to supply authenticated cultures to bona fide scientists, on request, promptly and without restriction on their ultimate use. The supply of certain bacteria such as pathogens (plant, animal or human) or patent strains will, however, be subject to statutory regulations and collections may impose further conditions. These may include, for example, proof that the requesting scientist holds the appropriate licence or permit to work with the cultures requested; that the request or order for cultures bears an authorised signature; and that an appropriate import licence is held. Supply of cultures to and from different countries, if pathogenic to man or animals, is subject to the International Air Transport Association (IATA) regulations whether sent by post or by air-freight. While the culture collection will effect the despatch of cultures as quickly as possible, delay can occur if those requesting the cultures are unaware of, or attempt to ignore, regulations. The collections, however, can only operate within the regulations. 3.1.2

Location of strains The primary information about cultures available from a particular collection will be found in its printed catalogue. These, however, are not published as frequently as, say, catalogues of commercial suppliers of chemicals or equipment and so are never fully upto-date. Many catalogues are now held on computers, and this enables quick and continual up-date by the collection and, in some cases, can be made available on-line (see Chapter 2). 49

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Catalogues are generally arranged alphabetically by genus and species names and while there is responsibility on collections to keep nomenclature up-to-date, the curators of most bacterial collections generally include also names which are invalid under the Bacteriological Code (Lapage et al., 1975) and the Approved Lists of Bacterial Names (Skerman et al., 1980), if only as cross-referenced entries. There are three reasons to justify such action. First, the Code itself specifically excludes collection catalogues as valid publications for naming bacteria (catalogues are considered to be ephemeral publications); secondly, the technically correct name under the Code may not be very familiar, or known at all, to the user and the catalogue is simply a device to help the user find the wanted strain; thirdly, some strains will be accessioned bearing names that are either unpublished or invalidly published, but which are not known by any other name. Collection catalogues can quite legitimately include invalid names, but these should be distinguished from valid names by some printing device such as a different typeface. Because catalogues become dated, they should not be used as guides to correct nomenclature. In the case of bacteria (and, incidentally, only in the case of bacteria), correct, valid nomenclature is readily available to the scientist by simple referral to the Approved Lists of Bacterial Names (Skerman et al., 1980) and subsequent Validation Lists published in the International Journal of Systematic Bacteriology. Printed catalogues often include tabulations of appropriate cultures for specified uses or applications, but do not include all the test result data on each strain held in the collection and it is not usually possible to carry out property-orientated searches for appropriate strains. As there is now a recognised need for such a facility, property-orientated computer databases are being developed (see Chapter 2). The larger service collections will also have cultures that are not listed in the catalogue. Deriving either from the collection's own research activity or from additional services such as identification, the collection may accumulate many further strains of species which are already represented in the catalogue in numbers adequate for most purposes. There may be, however, particular user needs where, for example, larger numbers of strains are statistically necessary. Supply of these uncatalogued strains will generally be subject to prior consultation with the curator. The user should bear in mind that catalogues, apart from their obvious function, serve as guides to the types of species held in the collection and that if a culture of a particular type or species is not listed

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in an available catalogue, yet would be expected to be included, then direct communication with the collection may be fruitful; if not, the collection will become alerted to an apparent deficiency. 3.1.3

Pricing policy The major service-supply collections receive funding from the public sector and levy fees for supplying cultures. The pricing policy adopted will not usually be determined solely by the collection but may be established in whole or in part by the parent organisation. Fees that correspond to full economic costs would mean unit costs far too high for what are re-usable (subcultured) products. The collections exist not only to supply cultures, but also to conserve microbial genetic resources for the future, and therefore must also keep and bear the costs of strains of little current demand, that are commercially unattractive stock items. Fees should not discourage use of the collection. It is mistaken to believe a real financial saving will obtain either from managing without authenticated cultures or from obtaining them indirectly at second or third hand without cost. Little reflection is needed to appreciate the insidious rising costs of falling standards and declining work practices or the costly risk of possible invalidation of work through use of a culture that has become mixed, contaminated, wrongly labelled or in some way changed at an intermediary point. Even when received for no fee, a culture is still not gratis, since someone has paid for maintenance costs. In practice, service collections have to be cost-conscious, but endeavour to strike a balance between pricing policies (as far as the collection can influence these) which relate to definable measurable costs and which yield an income that is significant in relation to costs but which does not result in a declining effective use of the collection. It is common practice to levy a higher fee to commercial clients than to non-commercial institutes. There may be further charges for posting and packing, air-freight if necessary, any special documentation that is only rarely needed (e.g. Certificates of Origin, which have to be validated through Chambers of Commerce), and national taxes (such as VAT in the UK). 3.1.4

Dispatch No service collection can be fully informed on Import/Export regulations of countries other than its own. The burden of compliance, therefore, rests with the clients, to inform the collection of their posses-

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sion of appropriate licences or permits if these are necessary. Another consideration is whether or not the cultures requested are Infectious Substances as defined by paragraph 3.6.6 in IATA Dangerous Goods Regulations (IATA, 1988), namely 'those substances containing viable micro-organisms or their toxins which are known, or suspected, to cause disease in animals or humans'. If the cultures are, in the best scientific judgement, infectious substances, then they are automatically classed by IATA as Dangerous Goods. This, in turn, means that packaging must conform to defined standards and dispatched packages must be accompanied by specified documentation (two 'Shippers Declaration for Dangerous Goods' forms if by air-freight, one only if by post); such cultures are prohibited from carriage in personal luggage, or on the person. Dispatch may be by post only if both national post offices accept such material. Postal delivery is preferable not only because it is cheaper than air-freight, but also because it is a 'delivery to the door' service. If either or both national post offices do not accept infectious substances, then air-freight has to be used. Dispatch and delivery is to an airport for collection, and requires expert assistance for customs clearance. 3.1.5

Accession policy

The service collections actively add strains ('accessions') to their stocks, both to fulfil their role as conservationist depositories of microbial genetic resources for the future and to satisfy demands placed upon them for supply. Most collections are specialised in one or other of the main sub-disciplines and scientists wishing to deposit their cultures should always first consult with the collection staff. If the intended collection is not, in fact, the most appropriate, advice will be given as to which collection would be more suitable. Accessions may arise from the collections' own research, or from cultures sent to the collection for identification that are found to be interesting in some particular detail. They may be actively solicited by the collection from following the scientific literature (noting, for example, the designation of a type strain, or a strain for a specific, well-defined function); they may arise unsolicited, or they may form part of a formal procedure such as a patent deposit under the Budapest Treaty or under national patent law (see Chapter 6). Service collections, however, cannot be expected automatically to assume responsibility for large collections simply because they can no

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longer be maintained at the laboratory of origin. The World Federation for Culture Collections (WFCC) has a committee for 'endangered collections' that aims to evaluate and relocate important collections. Criteria have been developed for evaluating such collections and individual service collections will tend to follow much the same criteria. Therefore an early approach to the WFCC committee or a potential host collection is necessary. Good documentation will be expected, and perhaps even cost estimates made. Under the normal accession circumstances, the depositor will be involved at the beginning and the end of the process. Initially, the depositor supplies as much relevant information as practical to the collection, with the deposited culture. This will be contained on an Accession Form. When the collection has fully processed, checked, preserved and quality-controlled the culture, a sample will be returned to the depositor for his confirmation that the collection version of the strain is satisfactory. 3.1.6

Records Collections maintain detailed records on each culture held. The accession form mentioned above, and from which a catalogue entry will be made, will be supplemented by the collections' own characterisation tests, viability and purity checks, and, in the case of a supply collection, further records regarding stock levels, issues, and to whom these issues have been made. Copies of packing or advice notes and invoices will also be kept. In the long term, a collection could become overwhelmed by paperwork, but the use of such aids as microfilming, or processing requests for cultures by computer and thereby keeping records of despatch on computer files, can alleviate this problem. In the special case of human pathogens, collections may retain original documents concerning issues of cultures for some years. At the NCTC, UK, for example, documents are kept for five years, except for those which include Hazard Group 3 (see Section 3.2.2 below), which are kept indefinitely. Much of this record keeping is only of an administrative nature. Records such as accession forms and results of collection quality control tests, however, comprise a scientific resource. 3.1.7

Uniqueness of collection strains When a strain is accessioned to a collection, it will be given a unique catalogue number. Strains obtained from service collections are

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referred to in the literature by these unique numbers, and so become recognised as standards. Results of work carried out in different laboratories at different times on strains defined as the same by bearing the same unique catalogue numbers, are directly comparable if the cultures were obtained from the collection. If a collection strain is obtained by any route other than directly from the collection it is misleading to refer to it by its unique catalogue number as the collection strain, since 'strain drift', contamination or mislabelling may have occurred. At best it may be called a culture 'derived from . . .'. The service collection catalogue number is unique to the version of the strain available only from that collection. Important and widely used strains will very probably be available from several different collections. The individual and separate collections will list their own numbers as the principal ones, but will also quote equivalent catalogue numbers of the other collections. Major collections have stringent quality control procedures and, when accessioning strains from each other, go to great lengths to verify 'identity' of their separate versions of the same strain. Some equivalence quoted in catalogues can be taken with a high degree of probability as being correct. This cannot be said, however, of strains whose 'identity' derives from diverse routes and it cannot be overemphasised that the direct comparison, evaluation, and reproducibility of work can be assured only (so far as is possible in biological work) by use of authenticated cultures obtained directly from the collection, and thereby exploiting to advantage their uniqueness. 3.2

Safety

3.2.1

Introduction

Since the introduction of the Health and Safety at Work etc. Act (1974) in the UK and the Occupational Safety and Health Act 1970 in the USA, a spate of guidelines, regulations or recommendations has been produced pertaining to the handling of microorganisms in research and production establishments, hospitals and educational departments. There has been a conscious attempt to ensure that acceptable safety procedures are established and maintained at all places of work. It is both nonsensical and damaging to impose safety requirements which are out of all proportion to the risks which are acceptable in everyday life. It is also foolhardy to ignore or avoid taking precautions against realistic hazards which the handling of certain microorganisms

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presents, either because of real or potential pathogenicity to man (see Section 3.2.2 below), or because they have been genetically manipulated (see Section 3.2.3 below). In the UK the Health and Safety Executive offers an advisory service to the community in all matters of safety and has inspectoral powers to enforce the law in instances where the Health and Safety at Work etc. Act is breached. Culture collections have a very important role to play in the safety area as often they provide the raw materials required for projects to be initiated. If a collection includes hazardous cultures, the curator has a three-fold responsibility: towards the collection's own staff and inhouse practices; towards the staff of delivery and transportation services; and towards the staff of the receiving laboratory. Distribution of materials represents a large proportion of a culture collection's activities, and must address all the problems associated with transportation by air, postal services or road, nationally or internationally. There is an ever increasing demand for microorganisms, animal or plant cell lines and genetically manipulated material of guaranteed pedigree, all of which have to be provided by the culture collections. The manner in which microbiological agents and cell lines can be handled safely is outlined in the following paragraphs. 3.2.2

Categories of pathogens

The latest guidance in the UK on the categorisation of pathogens is to be found in a report produced by the Advisory Committee on Dangerous Pathogens (ACDP) 1984, Categorisation of Pathogens according to Hazard and Categories of Containment. Bacteria, Chlamydia, Rickett-

siae, mycoplasmas, fungi, viruses and parasites are clearly categorised according to the hazard they present to workers and the community and four hazard groups are identified. Information is given on the degree of containment and protective clothing which should be applied during the handling of such organisms in the laboratory, including requirements for containment of infected animals. Hazard Group 1 are organisms that are most unlikely to cause human disease; Hazard Group 2, those that may cause human disease and which might be a hazard to laboratory workers but are unlikely to spread in the community; Hazard Group 3, those that may cause severe human disease and present a serious hazard to laboratory workers, and may present a risk of spread in the community but for which there is usually effective prophylaxis or treatment available; and,

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finally (and currently restricted to a few viruses only, e.g. Lassa fever virus), Hazard Group 4, as Group 3 but greater risks and usually no effective prophylaxis or treatment available. The majority of human pathogens are categorised as Hazard Group 2, and are considered safe to handle under normal open bench conditions providing good microbiological practices are observed. Hazard Group 3 pathogens must be handled only in protective cabinets and only by expert and designated staff. Table 3.1 lists the currently recognised Hazard Groups 2 and 3, by species names. For some species, further recommendations are made, such as required or recommended vaccination. Table 3.1. Hazard Groups 2 and 3 Bacteria, Chlamydia, Rickettsiae and Mycoplasmas (UK categorisation Advisory Committee on Dangerous Pathogens, 1984), with annotations ofATCC-PHS categorisation when different (ATCC, 1986). Note: in general UK Hazard Group 2 = ATCC-PHS Class II, and Group 3 = Class III; annotations are given below only when different for given species Genus/species

UK Hazard Group

Acinetobacter calcoaceticus Acinetobacter Iwoffi 'Actinobacillus actinoides' Actinobacillus actinomycetemcomitans Actinobacillus lignieresii Actinobacillus suis Actinomadura spp. Actinomyces bovis Actinomyces israelii Aeromonas hydrophila Arizona spp. Bacillus anthracis Bacillus cereus Bacterionemia matruchottii Bartonella bacilliformis Bordetella parapertussis Bordetella pertussis Borrelia (all spp.) Brucella spp. Campylobacter spp. Cardiobacterium hominis Chlamydia psittaci (avian strains only) Chlamydia (other strains) Clostridium botulinum Clostridium tetani Clostridium other spp. (except those known to be non-pathogenic)

2 2 2 2 2 2 2 2 2 2 2 3(v)

2 2 2 2 2 2 3 2 2 3

j(ATCC/PHS Class II or IV)a

2(v) (ATCC/PHS Class III)

2V 2

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Table 3.1. (cont.) Genus/species

UK Hazard Group

Corynebacterium diphtheriae 'Corynebacterium haemolyticum' 'Corynebacterium minutissimum' 'Corynebacterium ulcerans' Coxiella burnetii Edwardsiella tarda Eikenella corrodens Enterobacter aerogenes Erypsipelothrix rhusiopathiae ('insidiosa') Escherichia coli (except genetically crippled strains) Flavobacteriutn meningosepticum Francisella tularensis (Type A) Francisella tularensis (Type B) Haemophilus spp. 'Kingella kingae' Klebsiella spp. Legionellaceae Leptospira interrogans ('ictero-haemorrhagiae') all serovars Listeria monocytogenes Moraxella spp. Mycobacterium africanum Mycobacterium avium Mycobacterium bovis (excluding BCG strain) Mycobacterium bovis (BCG strain) Mycobacterium chelonei Mycobacterium fortuitum Mycobacterium intracellulare Mycobacterium kansasii Mycobacterium leprae Mycobacterium malmoense Mycobacterium marinum Mycobacterium scrofulaceum Mycobacterium simiae Mycobacterium szulgai Mycobacterium tuberculosis Mycobacterium ulcerans Mycobacterium xenopi Mycoplasma pneumoniae Neisseria spp. Nocardia brasiliensis Nocardia brasiliensis 'Noguchia granulosis' Pasteurella spp. Plesiomonas shigelloides

2V 2 2 2 3 (ATCC/PHS Class III or IV) 2 2 2 2 2 2 3 1 (v) (ATCC/PHS Class II, i 2 J reference to Types A, B) 2 2 2 2 2G 2 2 3V (ATCC/PHS Class II) 2 (ATCC/PHS Class III) 3V 2 2 2 2 (ATCC/PHS Class II) 3 (ATCC/PHS Class II) 3 (ATCC/PHS Class II) 3 (ATCC/PHS Class II) 2 2 3 (ATCC/PHS Class II) 3 (ATCC/PHS Class II) 3V 2 3 (ATCC/PHS Class II) 2 2 2 2 2 2 2

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Table 3.1. (cont.) Genus/species

UK Hazard Group

Proteus spp.

2 2

Providencia spp. Pseudomonas mallei Pseudomonas pseudomallei

Pseudmonas (other spp.) Rickettsia akari Rickettsia Canada Rickettsia conorii

3 (ATCC/PHS Class IV) 3 (ATCC/PHS Class IV) 2 3 3

3 (ATCC/PHS Class IV)

'Rickettsia montana' 'Rickettsia mooseri'

3 3

Rickettsia prowazeki Rickettsia rickettsii Rickettsia sennetsu Rickettsia tsutsugamushi Rochalimaea quintana

3 (ATCC/PHS Class IV)

Vole rickettsia 'Salmonella paratyphi A' Salmonella typhi

Salmonella (other spp.) Serratia liquefaciens Serratia marcescens Shigella dysenteriae (Type 1)

Shigella (other spp.) Staphylococcus aureus Streptobacillus moniliformis

3

3 (ATCC/PHS Class IV) 3 (ATCC/PHS Class III or IV)a 3 3

3 (ATCC/PHS Class II) 3v (ATCC/PHS Class II) 2 2 2

3 (ATCC/PHS Class II) 2 2 2

Streptococcus spp. (Lancefield Groups A, 2 B, C, D, G) Streptococcus (other spp.) (except those 2 known to be non-pathogenic) Treponema pallidum Treponema pertenue Vibrio cholerae (incl. El Tor) Vibrio parahaemolyticus Yersinia enterocolitica Yersinia pseudotuberculosis subsp. pestis (Y. pestis) Yersinia pseudotuberculosis subsp. pseudotuberculosis

2G 2G

2 (ATCC/PHS Class III) 2 2 3v 1

Modifications to containment level in the model code of practice: G = gloves must be used V = vaccination required (unless known to be immune) v = vaccination recommended (unless known to be immune) (v) = vaccine available, use discretionary a = certain strains Class III, others Class IV.

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In the USA, a similar categorisation of pathogens has been drawn up (US Department of Health, Education and Welfare, 1974; see also ATCC, 1986), defining Classes I, the least hazardous, to IV, the most hazardous. In Table 3.1, differences between UK and US categorisation are noted. The American and British guidelines vary slightly but both have been accepted by the World Health Organisation. Other countries tend to adopt the systems used by the UK or the USA. 3.2.3

Genetically manipulated microorganisms The control of genetic manipulation experiments in the UK is the responsibility of the Advisory Committee on Genetic Manipulation using its own classification of experiments 1-4. Guidance is offered in a series of newsletters which are constantly updated. The American control system is somewhat more complicated but all federal agencies that fund research related to biotechnology adhere to the policy that research in this field must conform to the requirements of the coordinated framework such as the National Institutes of Health Recombinant DNA Guidelines. Other countries engaged on recombinant DNA techniques have local guidelines or utilise the American or British guidelines. 3.2.4

Codes of practice In the UK all working microbiological laboratories are required by law to operate under certain national or institutional codes of practice. In the generality of this requirement culture collections are no exception, but at the same time their very nature and function makes them special cases. In the UK, all working laboratories are required by law to observe codes of practice, and the National Collection of Type Cultures (NCTC), for example, being a constituent part of the Public Health Laboratory Service (PHLS) is bound also by the Code of Practice governing all PHLS laboratories (Anon., 1988). This Code of Practice is concerned with general standards of work involving human pathogens. But the NCTC is also, perhaps, unique among PHLS laboratories with regard to at least the wide range of species handled within its laboratories, bulk-batching of these by freeze-drying, and in daily dispatching of cultures. As a supplement, then, to the general PHLS Code of Practice, the NCTC also has its own Code, to control the special functions.

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3.2.5

Conditions of supply of cultures

Reputable culture collections are aware of the responsibilities associated with the supply of cultures and require bona fide signatories before releasing hazardous pathogens. Some collections issue a leaflet to prospective recipients explaining that all cultures supplied by them must be regarded as potentially pathogenic and be handled by or under the supervision of competent persons trained in microbiological techniques. This includes compliance with national or local codes of practice. A leaflet issued by the NCTC, for example, sets the following conditions of supply: (1) that the recipient has read the warning; (2) that the cultures will be used by the recipient, or under his/her direct personal supervision, or handled only by persons authorised by the recipient; (3) that the cultures or derived subcultures will not be distributed to any person who has not read the warning; (4) that certain cultures, considered to present a special hazard are subject to further conditions specified in Conditions of Supply of NCTC Cultures: Hazardous Pathogens. All species

categorised as Group 3 pathogens fall into this group. NCTC also states that cultures within this group are supplied only in response to a request signed by a Head of Department (or persons authorised by him/her) and whose signature(s) are deposited on the appropriate form with NCTC. Advice is given to suggest that species within this category would not be requested unless the recipient's laboratory facilities and staff training conform to national or local codes of practice. NCTC suggests that further guidance can be obtained in the UK from the Health and Safety Executive (HSE) or Ministry of Agriculture, Fisheries and Food (MAFF). The same conditions apply to some species within the Group 2 listed organisms which NCTC consider require the same safe handling procedure as for those in Group 3. (5) that the responsibility for ensuring safe handling of the cultures after receipt rests with the recipient. In the USA the American Type Culture Collection (ATCC) requires evidence that the recipient is trained in microbiology and has access to a properly equipped laboratory with the appropriate containment facilities. Requests for Class III pathogens (US categorisation) must be

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accompanied with a signed statement assuming all risks and responsibility for subsequent use. Similar procedures operate in other countries and can be obtained from the major national collections. 3.2.6

Transportation of cultures

It is obvious that distribution of cultures, both domestically and internationally, is essential to further scientific endeavour worldwide and this entails use of public transport facilities. This inevitably means that non-scientists are involved who need to be convinced that all necessary safety measures are taken, and that safety aspects have been sufficiently thought out and discussed between the scientists and transport authorities before transportation of cultures is made. Culture collections are duty bound to adhere in every detail to published regulations. 3.3

References Advisory Committee on Dangerous Pathogens (1984). Categorisation of Pathogens according to hazard and categories of containment. London: HMSO. ISBN 011883761 3. American Type Culture Collection (1986). Packaging and Shipping of Biological Materials at ATCC. ATCC. ISBN 0-930009-14-2. Anon. (1988). Safety Precautions. Notes for Guidance, 3rd edn. PHLS. ISSN 0144-1264. Health (1988). Dangerous Goods Regulations, 29th edn. International Air Transport Association, Montreal and Geneva. ISBN 92-9035-109-8. (Note: new editions are produced each year.) Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D., Seeliger, H. R. R. & Clark, W. A. (1975). International Code of Nomenclature of Bacteria, 1976 Revision. ASM. ISBN 0-914826-04-2. Skerman, V. B. D., McGowan, V. & Sneath, P. H. A. (1980). Approved lists of Bacterial Names. International Journal of Systematic Bacteriology, 30, 225-420. (Note: reprinted in hard-back book form by ASM, 1980. Corrigenda published by Hill, L. R., Skerman, V. B. D. & Sneath, P. H. A. (1984). International Journal of Systematic Bacteriology, 34, 508-11.) US Department of Health, Education and Welfare (1974). Classification of Etiologic Agents on the Basis of Hazard, 4th edn. Centers for Disease Control, Atlanta, Georgia 30333, USA PHS.

4 Culture and maintenance L. R. HILL, M. KOCUR and K. A. MALIK

4.1

Culture The variety of nutritional requirements covering the whole spectrum of known bacteria is wide, and aspects of initial culture of bacteria that will be considered here will be restricted to those relating to the subsequent process of diverse preservation techniques. Reference should be made to standard textbooks for information on how best to grow different species, details of media formulations, pH, gaseous conditions and optimum incubation temperatures and times. However, a number of general factors must be borne in mind regarding culture for preservation. 4.1.1

Primary isolation An obvious first requirement is to ensure that the culture is pure. Wherever practicable, the use of solid media is to be preferred to liquid media, since these allow plating out and subsequent singlecolony isolations. In medical bacteriology, one plating out and singlecolony isolation is usual for immediate investigations (for example, identification or determination of antibiotic sensitivity), where speed of obtaining an answer is the over-riding factor. For less urgent requirements and for preservation, however, it is advisable to go through two or even three successive platings out and single-colony isolations to ensure purity of the culture.

4.1.2

Enrichment Primary isolation may sometimes be preceded by enrichment of the source material and usually this will be done in liquid media. Indeed, liquid media may be essential if the required bacterium 62

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requires, for example, good aeration or fluxing with special gases. Plating out from appropriate dilutions will yield single colonies and again, wherever practicable, further culture should be carried out on solid media. 4.1.3

Selective media Primary isolations are often made on selective media, especially in medical bacteriology. These media include inhibitors of undesired species, but it must be remembered that often the inhibitors are bacteriostatic and not bacteriocidal. Thus a single colony of an apparently pure culture may still harbour some viable cells of a contaminant. For preservation purposes, it is imperative that wherever selective media have been used, there should follow a single-colony subculture onto a general, non-inhibitory medium. 4.1.4

Retention of purity check plates For many practical applications, bacteria are grown for their optimal period of time and culture plates are then discarded. For preservation purposes, it is advisable to retain plated-out cultures for longer periods and to re-examine these systematically for any evidence of slow growing contaminants still present. 4.1.5

Multiple colony cultures There is one exception to the standard single-colony-pick technique which arises when there is a need to replenish stocks of an already preserved culture. The pre-existing preserved culture should again be plated for single colonies, but once purity has been ensured further subculture should be made from several single colonies to form one new culture. This avoids the risk of selecting a mutant colony of the original culture, which could result from a single colony isolation; if this occurred then the replenished stock would not be identical with the original. 4.1.6

Colony variation Certain species of bacteria exhibit variation in colony morphology to a degree that causes doubt about whether it is a pure culture. Clearly, such cultures may require many more platings out and single-colony-picks to establish that the variation is a permanent, genetically inherent feature. The phenomenon of 'sectored' colonies, more noticeable in pigmented species, is usually less problematical,

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L. R. Hill, M. Kocur and K. A. Malik

since it occurs in isolated colonies, and subcultures from sectors often yield further sectored colonies. In all cases of colony variation, time and effort expended on extra single-colony subculture is well spent. 4.1.7

Pre-preservation culture The stages of checking that a culture is pure will have been made on Petri dish cultures. The preparation of a culture for preservation is best done, however, from a final slope culture. Harvesting colonies from a dish is not to be recommended as it risks introduction of aerial contaminants. Suspensions of cells are more easily made from slope cultures, using Pasteur pipettes and liquid medium to form a cell suspension. 4.1.8

Checking suspensions Suspensions of cells ultimately used in the preservation process should themselves be checked after dispensing, and this final quality control step is carried out by placing a drop of the suspension onto a non-selective, non-inhibitory medium and to plating out, incubating and re-examining for purity. 4.1.9

Optimum growth Poor initial growth of a culture is often the main cause for apparent failures in preservation techniques. Generally, late logarithmic phase cultures lead to better survival rates but, whenever possible, the particular requirements of each individual culture should be met. In practice, this target may be more easily achieved in the small specialised collection where the microbiologist may have a greater knowledge of optimum growth media and conditions for individual strains. In large collections, and especially service supply collections, a compromise has to be made between best methods for individual species (or even strains) on the one hand and standard methods for efficient scheduling of work on the other hand, in order to obtain good results with most cultures, most of the time. In striking such a balance, however, a significant proportion of time and effort will need to be paid to a minority of cultures with known special requirements. 4.2 4.2.1

Maintenance Active, or serial, subculture Many, probably most, bacteria can be maintained by active growth on appropriate media and periodic transfer to fresh media. The

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culture preserved in this way is maintained by alternate cycles of active growth and storage periods obtained by series of subcultures. Whether active subculture is convenient, or even secure, for long-term maintenance is, however, less certain. Subculture is a familiar technique to all practising microbiologists; it calls for basic skills of aseptic technique, but no special equipment. If many cultures are to be maintained actively, then steps need to be taken to prolong the period of time elapsing between subcultures. A rich medium giving good growth of a particular species may not necessarily be the best for maintenance. A poorer medium may give slower growth so that the culture will take longer to reach stationary phase, and less frequent subculture will be required. Several factors need to be borne in mind. Whenever possible, solid media should be chosen in preference to liquid, as contaminants can be more readily observed. Stab, rather than slope, cultures are often used for maintenance, but there do not appear to be published data to show that this is more successful. Oxygen-sensitive bacteria may benefit from stab culture, if only as an extra safeguard when subculturing at the bench. Rich media may mean not only more frequent subculture, but possibly also quicker accumulation of toxic end-products of metabolism, with the risk that a stationary phase culture may quickly become non-viable. Media should be dispensed in impervious containers that can be securely sealed. Glass 6 ml bottles, with rubber-lined, metal screw-caps are widely used. Organisms with somewhat fastidious oxygen requirements may be grown with the caps deliberately loosened but, once growth is obtained, the caps are tightened again for storage purposes. Cotton wool plugged tubes are not adequate, as media will quickly dry out and cultures will be lost. Other methods of sealing such as waxed corks, rubber bungs, even waxed cotton wool plugs are rapidly disappearing from bacteriology, since for active subculture maintenance methods they are very inconvenient. The time period appropriate for subculture is a matter of experience and will vary according to a combination of the inherent robustness or sensitivity of the cultures, the media used, and the effectiveness of the seals. The greater the variety of species in a collection to be maintained by active subculture, the greater becomes the importance of setting up suitable protocols to ensure which cultures should be subcultured and at which time. Often frequency of subculture can be reduced simply by transferring grown cultures to 4 °C in a refrigerator, so reducing the

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L. R. Hill, M. Kocur and K. A. Malik

metabolic rates of the cells. Some delicate species (e.g. Neisseria gonorrhoeae) will immediately die following this treatment. Overlaying grown cultures of non-anaerobes with sterile medicinal-grade liquid paraffin excludes oxygen, and may also reduce metabolic rates. With any such devices, and even with prior knowledge, it is advisable to maintain parallel cultures at room temperature and non-overlaid, until the success of the method has been assured for particular cultures. Active subculture as a maintenance method is hazardous, inconvenient and possibly unsound. These disadvantages may be kept within reasonable limits with a small collection, but become compounded the larger the collection becomes, either in numbers or in variety of cultures, or both. Human error hazards of mislabelling, non-adherence to subculturing protocols, technical incompetence, accidental introduction of aerial contaminants are not exclusive to active subculture but at the same time - and given the tedious nature of the technique - are more probable. Inconvenience derives from active subculture being labour-intensive and, for a supply collection, the bulk storage of replicates of subcultures is demanding on space. Additionally, delivery of cultures in response to requests becomes slow if the practice is to make a subculture at the time of request. Maintenance of cultures aims not only to keep cultures viable and pure, but also to ensure their properties remain unchanged. Active subculture may well be scientifically unsound in this respect, for in repeated cycles of active metabolism the chances of genetic drift increase.

4.2.2

Drying, or desiccation; L-drying

Dehydration reduces the metabolic rates of organisms, but not all species survive simple drying methods without added protective agents. Soil, sand, kieselguhr and silica gel have all been used successfully as substrates. These substances are distributed into receptacles that permit eventual air-tight sealing, drying and sterilisation. Drops of suspensions of cells are then added and allowed to dry at room temperature. When dry, the receptacle is sealed, for the substrate is hygroscopic. Paper discs or strips, contained in vials or laid out in Petri dishes, or plugs of substances such as peptone, starch, dextrin, or tufts of cellulose or quartz wool (Annear, 1962) have all been used in much the same way. If the drying process is accelerated by applying a vacuum

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(e.g. by transferring the vials or Petri dishes to a vacuum-desiccator or attachment to freeze-drying apparatus, see below), then the process is generally called 'L-drying', that is, drying from the liquid state. Drying under vacuum can be manipulated so that no freezing occurs; this is in contrast to lyophilisation (see Section 4.2.4, below) in which sublimation - drying from the frozen state - occurs. In practice freezing can be avoided by several means. One method is to use small volumes of cell suspension spread over large surface areas. It is understood that application of a vacuum then leads to quick drying before the liquid freezes. This is probably what happens in most simple drying procedures. L-drying is used less commonly than lyophilisation as it needs much time and continued care. The method has recently been improved and simplified (Malik, 1988a) and is used at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM) for the preservation of various sensitive bacteria which otherwise are damaged by freezing or freeze-drying and fail to survive. In this method activated charcoal is used together with a protective agent, and serves as a good carrier material. In contrast to conventionally used carriers, such as cellulose fibre which is a bad heat conductor, activated charcoal is an excellent thermal conductor. It ensures proper distribution of heat in the samples, thereby minimising the possibility of freezing. In combination with other additives, activated charcoal serves as an ideal adsorption base and almost no frothing occurs, even during rapid evacuation and degassing. During L-drying under reduced conditions (Malik, 1988a), activated charcoal is a neutral and harmless absorbant, important for cultures that are sensitive to some toxic reducing agents. Several anoxygenie light-sensitive phototrophic bacteria have been dried successfully in the presence of activated charcoal which, by absorbing light and oxygen, is effective against photo-oxidation. Although the use of protective agents increases the survival and stability of microorganisms during L-drying, harmful effects have been reported due to the presence of certain additives (thiourea, cysteine, sodium sulphide, etc.) in the rehydration medium during the resuscitation process. A suitable rehydration medium improves survival and is reported to be involved in the repair mechanism of DNA damage which results during drying. The presence of activated charcoal in the dehydration medium proved harmless and rather helpful during resuscitation by adsorbing various harmful radicals (superoxide and other free radicals created by various electron donors in reaction with oxygen).

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L. R. Hill, M. Kocur and K. A. Malik

For drying very sensitive anaerobic bacteria, small gas-tight screwcap glass ampoules with butyl rubber septa are used and L-drying is carried out under gas-tight conditions using activated charcoal as reductant. After L-drying, the vacuum is replaced with sterile argon or nitrogen gas. Complete anaerobiosis is secured without the use of anaerobic chamber or glove boxes. For reactivation, reduced medium is injected through the rubber septum into the dried cell suspension, without opening or cracking the ampoules. Simple drying methods are probably improved by addition of protective agents, such as skim milk or 10% sodium glutamate, in the cell suspension, but survival is probably governed most by the intrinsic nature of the organism - for example spore-formers survive well. Gelatin discs are used commercially. Originally described by Stamp (1947), thick suspensions of bacteria in nutrient broth are added to melted nutrient gelatin at 30 °C. Drops of the gelatin suspension are dispensed onto waxed paper in a Petri dish, which is then transferred to an evacuation desiccator. Dried gelatin discs are finally transferred aseptically to a sterile, screw-cap 6 ml bottle, which is then sealed. 4.2.3

Freezing

Freezing is a common process for storage of bacteria. Thus, thick bacterial suspensions can be frozen at a temperature of —30 °C, which can easily be attained with commercially available deepfreezers, although lower temperatures than these are preferable (see below). Metabolic rates are reduced by lowering the temperature and in the extreme case of storage in liquid nitrogen at —196 °C, are considered to be reduced to nil. However, cryopreservation is not without difficulties. Freezing and thawing constitute a well-known technique for actually disrupting cells. Moreover, as water is removed during freezing as ice, electrolytes become increasingly concentrated in unfrozen water, and this too may be harmful, since electrolyte concentrations outside cells become very different from those inside cells, leading to osmotic stress. Lowering the temperature to below the eutectic point leads to the whole suspension becoming frozen. Cryoinjury is thus a complex subject, involving electrolytic effects, ice formation with damage to membranes, and 'removal' of water from intracellular proteins, nucleic acids, and other cellular components. Nevertheless, the storage of grown cultures in deep-freezers is successfully practised for a very wide range of cell types. Lapage & Red-

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way (1974) gave a summary of precautions to take to reduce cryoinjury; the main ones are to keep electrolytes in the suspension to a minimum and to add protective agents such as glycerol or dimethylsulphoxide (DMSO). The common deep-freezers, at -20 to -30 °C, provide possibly the worst storage conditions, as eutectic mixtures of water and electrolytes are formed down to these temperatures. However, mechanical freezers are now available which operate at —70, —80, or even —135 °C, though capital costs become greater as the storage temperature is decreased. Ultra-low temperature preservation (cryopreservation) has a long tradition with certain kinds of living cells (e.g. semen preservation in animal breeding) and is becoming increasingly popular for the preservation of microorganisms, cells and tissues. Microorganisms sensitive to freeze-drying can be preserved in liquid nitrogen. With controlled freezing and thawing it is possible to freeze-store microbes with good genetic stability and viability (Bridges, 1966; Daily & Higgins, 1973; Kirsop & Snell, 1984). The greater efficiency in recovery of the bacterial cells from the frozen state is important as it minimises the possibility of the selection of cells resistant to the technical manipulations employed during the process of cryopreservation. With liquid nitrogen storage, the rate of cooling is important, but differs for different biological materials (for example, the optimum freezing rate for unprotected human red blood cells is about 3000 °C min" 1 , for yeast cells 10 °C min ~1, for bacteria between 1 and 5 °C min" 1 ). This phase can be controlled, and test runs made by the use of programmable freezers (Planer, Cryo-med) or two-stage freezers (Linde BF-5). In practice a relatively slow initial cooling rate can be obtained by keeping cultures in the neck of the liquid nitrogen storage unit for some minutes and then, after freezing, lowering containers into it. It is not good practice to plunge cultures directly into liquid nitrogen. For safety reasons, cultures are better stored in the vapour phase above the liquid nitrogen itself, since if containers are immersed, then liquid nitrogen may seep into any imperfectly sealed capillaries, ampoules, or vials containing the bacterial suspensions: then, on removal from storage the nitrogen will instantly change to the gaseous phase and an explosion may follow. However, storage in the vapour-phase provides temperatures that may be suboptimal, possibly above —135 °C (at which temperature recrystallisation of ice occurs), and may fluctuate. This may not be critical for many cultures, but molecular stability

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L. R. Hill, M. Kocur and K. A. Malik

becomes increasingly important for genetically manipulated strains for biotechnological application. For non-pathogenic bacteria, polypropylene drinking straws can be used as primary containers. The straws are cut into short lengths and held close to a flame for one end to melt and seal; they are then sterilised by autoclaving and filled aseptically with the bacterial suspension. The open end is then sealed again by holding close to a flame. Five or six such straws can be accommodated in a cryotube. For pathogenic bacteria, a more reliable seal is needed and, to achieve the same economy of storage space and replicates of the same culture, glass capillary tubes can be used, sealing both ends in a flame after having first taken up a minute volume of suspension by capillary action. It is generally found that the best recovery of cultures from liquid nitrogen storage is achieved by quick thawing. However, the events during thawing are complex and it is not only a simple reversion of the freezing process. Slow warming rates can lead to ice crystal formation, whereas a rapid warming at about 100 °C min" 1 generally will give better survival since ice crystal damage is minimised. To achieve rapid warming rates the samples (cryotubes) removed from liquid nitrogen are quickly immersed into water baths at 37 °C. For protection of cells from freeze-damage, cryoprotective agents should be used. Cryoprotectants belong to two main classes, defined on the basis of their permeability to cells. The penetrating compounds such as dimethylsulphoxide (DMSO), glycerol, methanol and ethylene glycol are low molecular weight compounds and are generally applied in high concentrations (0.5-1.5 M). The non-penetrating agents such as hydroxyethyl starch (HES), polyvinyl pyrolidone (PVP), sugars, proteins and polyethylene glycol are used at much lower concentrations (0.01 M), as toxicity at higher concentrations may cause problems. All these substances have a high affinity for water and are soluble even at low temperatures, and this may relate to their protective effects, although their exact mode of action is not known. Proposed mechanisms include effects on ice crystallisation, lowering of freezing points, increasing membrane permeability and stabilisation of cellular structures. Penetration of the cryoprotectant into the cells is not always necessary, as indicated by the effectiveness of high molecular weight substances PVP or HES. However, successful cryoprotection is the result of all components acting effectively inside and outside the cells. For cryopreservation of living material, many different protocols

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have been published varying with special needs, personal preferences of materials or the type of organism under study. For the purpose of a culture collection, several prerequisites are necessary, which include a minimum of storage space, applicability to a wide range of different organisms, and good survival over storage periods of several years. Several new miniaturised and economical methods have been developed, which reduce the volume of the cell suspension to be preserved and of the unit holding this. Small glass beads, mini-ampoules (Feltham etal., 1978; Malik, 1984, 1985) or glass capillary tubes are used to prepare several samples from the same batch of the cells (Dietz, 1975; Hippe, 1984; Hippe, Hoffman & Malik, 1986). Storage of large numbers of cultures in liquid nitrogen units requires proper organisation within the storage unit and manufacturers provide inventory racking systems that can be successfully used. 4.2.3A Cryopreservation of anaerobic bacteria

The technical details of the method used at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM) (Malik, 1984), for cryopreservation under anaerobic conditions are described below (see also Fig. 4.1). Ampoules: neutral glass screw-cap (10 X 30 mm) of 2 ml capacity (Varian GmbH, Darmstadt, FRG). The ampoules are provided with rubber septa and plastic screw-caps with holes for injection of samples. For strict anaerobes the red rubber septa are replaced with oxygen-impermeable butyl rubber septa. The ampoules are washed, rinsed with distilled water, tightly closed and autoclaved. Before use these are labelled with the collection number of the strains to be preserved. For freezing in liquid nitrogen aluminium canes or holders are used, to which the ampoules are clamped. Cryoprotective agent: dimethylsulphoxide (DMSO) solution (20% v/v in H2O) is sterilised undiluted at 114 °C for 10 min or is filter sterilised. Other suitable protective agents recommended in the literature can also be used. Preparation of cell suspension: anaerobic cultures are grown under appropriate conditions until mid- to late- logarithmic phase of growth. For harvesting, the cultures are centrifuged for 30 min at 4000 x g in the screw-cap bottles in which cultures are grown. The supernatant is

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cotton-filled syringe

J

Mini water bath at 37° C

Removal of inoculum

Removal of supernatant for a thick cell suspension or a pellet

Asceptic evacuation of mini screw-cap glass ampoules

Ampoules for refreezing into Liquid Nitrogen

Liquid Nitrogen container

Ampoules clamped on to an aluminium cane

Fig. 4.1. Cryopreservation under anaerobic conditions.

± f

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removed anaerobically under a stream of nitrogen gas using an overflow butyl rubber tube of about 5 mm diameter with Luer Lock adaptors at both ends and fitted with long syringe needles of 10-15 cm length (see Fig. 4.1 A). To obtain sterile nitrogen gas, a sterile cottonfilled syringe is attached to a conduit connected to the N 2 gas (99.99%) cylinder. The pellet is resuspended carefully in ice-cold sterile DMSO solution (5% v/v in H2O). In the case of halophilic strains or cells which do not form a pellet, a thick bacterial suspension (in growth medium) is mixed in the ratio 3:1 with ice-cold sterile DMSO (20% v/v in H2O). The cells are allowed to equilibrate with the cryoprotectant for 15 min in an ice bath. Filling of ampoules: using a sterile gas-tight 5-10 ml syringe, the ampoules are evacuated for anaerobiosis and to facilitate filling (Fig. 4.1B). 1.5 ml of cell suspension (108-1010 cells ml"1) is withdrawn with a sterile oxygen free syringe (already flushed with nitrogen gas) and injected into each ampoule (Fig. 4.1C). The ampoules are immediately clamped onto a labelled aluminium cane, placed in canisters and frozen (Fig. 4.ID). Revival of cultures: thawing of ampoules is by immersion to the neck in a water bath at 37 °C (Fig. 4.IE). After thawing, the outer surface of the ampoules is dried by wiping, the septum is flame sterilised and with a 1 ml oxygen-free syringe, a small volume (about 0.05 ml) of inoculum is withdrawn and injected into 5-10 ml liquid growth medium (Fig. 4.IF). The rest of the cell suspension is immediately frozen again (a wax block rack, chilled to —30 °C, is used for transportation to the liquid nitrogen container, see Fig. 4.1G) in liquid nitrogen for later use. The DMSO, which is often toxic during growth, is diluted 100-200 times in the culture medium to a non-inhibitory concentration. The inoculated growth medium is incubated under appropriate growth conditions. Estimation of viability counts: for the estimation of viable cell counts, 0.5 ml of inoculum is transferred from the unfrozen and from the thawed cell suspension into pre-reduced 4.5 ml medium in screw-cap tubes (Hungate tubes with septa, Bellco Glass Inc., 2047-16125) and 6-8 serial decimal dilutions are prepared using oxygen-free syringes. Agar roll tubes can be prepared for viable colony counts if such facilities are available. Alternatively counts on agar plates can be performed in an anaerobic glove box or anaerobic jars. Single plates can be incubated

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anaerobically in anaerobic bags, such as Bio-bags (Type A, Marion Scientific Corporation, Kansas City, MI, USA). For cultures which are difficult to grow in, or on, agar only liquid dilution series are made and numbers of cells determined using the most probable number method (MPN). Disadvantages of liquid nitrogen storage are the relatively high costs for equipment and maintenance and the absolute need for a regular supply of liquid nitrogen, but these drawbacks are greatly compensated by the many advantages. Freezing as a maintenance method, whether by mechanical refrigerators or by liquid nitrogen, can lead to difficulties for large culture collections. For example, once stocks are in storage, they still need attention; refrigerators must be regularly serviced and, if possible, connected to emergency electrical sockets so that in the event of a power cut they continue to function. There have been occasions where whole stocks have been lost due to failure to top up liquid nitrogen storage units, or to close lids properly. Automatic top-up devices are worth the investment, although these also can fail. Even the most reliable supplier of liquid nitrogen may be subject to industrial disputes that prevent delivery, and collections should register with suppliers for priority deliveries. The above difficulties should be weighed against the advantages of ultra-low temperature storage, in terms of molecular stability. If the latter is of paramount importance (for example, for the biotechnological use of constructed strains), then stability should take precedence over convenience. 4.2.4

Freeze-drying, or lyophilisation

This is probably the most widely used technique for maintaining bacterial cultures. While the preparation and processing of stocks are both labour-intensive, with specialised skills demanded, and relatively expensive in capital equipment, once processed the stocks call for little further attention. If the initial freeze-drying has been successful, with high viability, long-term losses are very rare. The cultures are viable, but metabolically inactive; they are easily returned to active growth. Freeze-dried ampoules are convenient for despatch and delays in delivery (at airports, for example) usually do not affect them; ampoules are small enough to be packaged in a manner to protect intermediaries (see also Chapter 3). Very few bacterial genera

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fail to survive lyophilisation, and in these cases some other method of maintenance has to be used, such as liquid nitrogen storage; in some cases where poor results are obtained, attention to standard protocols could well resolve the problem. Freeze-drying is a multi-stage process; it begins with freezing, a temporary stop to metabolic activity, then continues with the removal of water without thawing (sublimation), and ends with a dried product which, if sealed either under vacuum or under an inert gas, can be stored at room temperature with no further metabolic activity until water and nutrients are restored. Freeze-drying machines are available with a wide range of capacities. Even the smallest in-house specialist collection will benefit from obtaining a small freeze-dryer, or gaining access to one. For successful lyophilisation, cells of the microorganisms to be lyophilised should be suspended in a medium for protection against known freezing and drying injuries. Protective agents are needed both to prevent intracellular ice crystal formation, total desiccation (which damages DNA and kills the cells) as well as to neutralise other harmful effects such as electrolytic effects and the presence of free radicals (Greaves, 1964; Lapage et al, 1970; Redway & Lapage, 1974; Malik, 1976, 1988b). A few effective protective agents such as skim milk (20% w/v) and glutamate (5% w/v) or meso-inositol (5% w/v) or honey (10% w/v) or raffinose (5% w/v) have been recommended by Malik, 1988b. Because it is such a widely used technique, there are many variations in technical detail. The UK National Collection of Type Cultures (NCTC) method described below is used for a medically important collection, that includes many fastidious or delicate species, and so provides a suitable model. 4.2.4A Method of lyophilisation suitable for a wide range of bacteria

[Many other methods are applicable and the reader is referred to the list of references on p.78.] Culture: incubate heavily inoculated solid, rich, sloped media to obtain late logarithmic, confluent growth. Suspension: harvest the growth by adding 1-2 ml suspending medium with a Pasteur pipette and gently wash off the growth with the pipette; emulsify to make a uniform suspension. Avoid creating aerosols.

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Suspending media: 5% inositol in nutrient broth (Oxoid) or serum (Wellcome) (Redway & Lapage, 1974) are used, but a great variety of media has been recommended in the literature. Ampoules: neutral glass, 100 X 7-7.5 mm ampoules (Edwards High Vacuum) are acid-washed in 2% HC1 overnight, thoroughly rinsed first in tap and finally distilled water and dried. Strips of blotting or filter paper, 5 x 30 mm, bearing the typed or stamped culture identification number are placed in the ampoules, which are then plugged with cotton wool and sterilised by autoclaving. Filling ampoules: approximately 0.1-0.2 ml of the bacterial suspension is delivered by Pasteur pipette to each ampoule. This is a relatively slow, yet skilled job; the sides of the ampoule must not be contaminated. Spin-freeze: ampoules are placed in the head of a centrifugal freezedryer and the vacuum is applied. Suspensions become frozen by the latent heat of evaporation under the reduced pressure; centrifugation prevents frothing of the suspensions which would otherwise arise from evolution of dissolved gases. After 10-15 min the centrifugation is terminated. The suspensions will be completely frozen and will remain so provided the vacuum is maintained. Primary drying: ampoules are left in the vacuum chamber for about 3 h. 90-95% of removable water is removed during this stage by sublimation and recondensation as ice in the refrigerator unit. Plugging and constricting: ampoules are removed, sterile cotton wool plugs inserted into each and pushed halfway down the ampoule with a rod. With a constricting machine, or by hand, ampoules are constricted between the top of the plug and the open end of the ampoule to produce a short capillary section of about 2 mm diameter. Secondary drying: constricted ampoules are attached to secondary-drying manifolds and a vacuum is applied. Further water is removed and trapped by chemical reaction of water vapour with phosphorus pentoxide. Vacuum is maintained overnight, after which residual water will be about 1%. Sealing: sealing takes place in situ on the manifolds, under vacuum.

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Each sealed ampoule is checked for vacuum with a high frequency spark tester prior to storage, and rechecked whenever withdrawn from storage. Special precautions for hazardous pathogens: the above procedure is hazardous at the centrifugal stage since aerosols may be created or breakages occur. This can be avoided very simply by prefreezing sloped ampoules in crushed, solid CO2. Suspensions are quickly frozen and are transferred to the vacuum chamber of the centrifugal freeze-dryer and the vacuum applied. No centrifugation is used. Optimising the freeze-drying protocol: generally freeze-drying is achieved by rapid freezing and subsequent drying. During some comparative studies it has been observed that even well-tried additives prove less effective during this process compared with slower freezing of the samples prior to drying. A new freeze-drying method has recently been reported in which technical details have been defined and optimised for the preservation of a comprehensive collection of nitrogen-fixing and other fragile bacteria (Malik, 1988b). The method is based on slow freezing of cell suspensions (about 1 °C min" 1 to about —30 °C) prior to prolonged primary freeze-drying. This method avoids rapid desiccation. This is followed by short (2-3 h) secondary drying.

4.3

Quality control Culture collection quality control should be as extensive as practicable and has three primary targets: checking that cultures issued are viable, pure, and authentic. All three elements should be checked at the time of storing stocks. With preservation methods such as active subculture the visible growth of strains in the next tube serves as the viability check, but it is not quantitative. With freeze-dried stocks, it is important to carry out a quantitative determination of colony forming units (CFUs) per ml of suspension prior to and immediately after freeze-drying. Viability levels recorded over many years of storage for a wide range of bacterial genera will be found in Lapage et ah (1970), Sourek (1974), and Rudge (1984). Viability, or other check plates of cultures, are examined for absence of contaminants but, unlike most bacteriological work, there is a priority in confirming the exclusion of slow growing contaminants in culture collection work. Viability plates and cultures plated out for

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single colonies are deliberately kept for longer periods and re-examined. Post-drying viability plates can be subcultured for checking the properties of cultures. The extent to which phenotypic characterisation is carried out by the culture collection will depend on its resources, the nature of the organisms and so on. However, it is self-evident that the value of a collection to the clients depends as much on the certainty that cultures requested are indeed authentic as that they are viable. 4.4

Records The conservation work of a culture collection depends not only on collecting representative, or special, cultures or in preserving them successfully, but also on keeping full and updated records. Thus original data supplied by the depositor must be recorded (source and origin of cultures; any special properties; dates of receipt); maintenance data (subculture, preservation, stock levels, records of viability checks); records of phenotypic test results or other characterisation records. The greater the amount of information purposefully recorded and regularly updated, the greater the value of the collection. 4.5

Security The value of a collection cannot easily be expressed in financial terms: nevertheless, the accumulated years of effort in collecting, preserving, and record keeping (not to mention the initial work carried out in numerous laboratories by depositors), deserve security and protection; this applies not only to service supply collections, but to any collection of valuable isolates. Total loss of a collection can be prevented by off-site storage of samples of each culture and copies of original documents relating to each culture. As computers become more widely used in culture collections, these too provide efficient back-up for the records. 4.6

References Annear, D. I. (1962). Recoveries of bacteria after drying cellulose fibres. Australian Journal of Experimental Biology and Medical Science 40, 1-8.

Bridges, B. A. (1966). Preservation of microorganisms at low temperature. Laboratory Practice 15, 418.

Daily, W. A. & Higgins, C. E. (1973). Preservation and storage of microorganisms in the gas phase of liquid nitrogen. Cryobiology 10, 364-7.

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Dietz, A. (1975). Nitrogen preservation of stock cultures of unicellar and filamentous microorganisms. In Round Table Conference on the Cryogenic Preservation of Cell Cultures, ed. A. P. Rinfert and B. Lasalle, pp. 22-36. Washington DC: National Academy of Sciences. Feltham, R. K. A., Power, A. K., Pell, P. A. & Sneath, P. H. A. (1978). A simple method for storage of bacteria at —76 °C. Journal of Applied Microbiology 44, 313-16. Greaves, R. I. N. (1964). Fundamental aspects of freeze-drying bacteria and living cells. In Aspects theoretiques et industriales de la lyophilization, ed. L. Rey, pp. 407-10. Paris: Hermann. Hippe, H. (1984). Maintenance of methanogenic bacteria. In Maintenance of Microorganisms: A Manual of Laboratory Methods, ed. B. Kirsop and J. J. S. Snell, pp. 69-81. London: Academic Press. Hippe, H., Hoffmann, P. & Malik, K. A. (1981). Capillary tube method for freeze preservation of microorganisms. In Fourth International Conference on Culture Collections, 1981, Brno, Czechoslovakia. Kirsop, B. & Snell, J. J. S. (ed.) (1984). Maintenance of Microorganisms: A Manual of Laboratory Methods. Academic Press, London. Lapage, S. P. & Redway, K. F. (1974). Preservation of bacteria with notes on other microorganisms. PHLS Monograph No. 7, London: HMSO. Lapage, S. P., Shelton, J. E., Mitchell, T. G. & Mackenzie, A. R. (1970). Culture collections and preservation of bacteria. In Methods in Microbiology, Vol. 3A, ed. J. R. Norris and D. W. Ribbons, pp. 135-228. London: Academic Press. Malik, K. A. (1976). Preservation of Knallgas bacteria. In Proceedings of the Fifth International Fermentation Symposium, ed. H. Dellway, pp. 180. Bonn and Berlin: Westkreuz Druckerei und Verlag. Malik, K. A. (1984). A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions. Journal of Microbial Methods 2, 41-7. Malik, K. A. (1985). Modern Methods of Gene Conservation. A Laboratory Manual. Pakistan Science and Technology Information Centre, Islamabad, Pakistan: PASTIC press. Malik, K. A. (1988a). A simplified L-drying method for the preservation of bacteria sensitive to freeze-drying. p. 74. In Proceedings of the Sixth International Congress of Culture Collections, Maryland, USA. Malik, K. A. (1988b). A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria. Journal of Microbial Methods 8, 259-71. Malik, K. A. & Claus, D. (1987). Bacterial Culture Collections: Their importance to Biotechnology and Microbiology. In The Biotechnology and Genetic Engineering Reviews, Vol. 5, G. E. Russel, ed. pp. 137-97. Ferndown, Dorset: Intercept Ltd. Redway, K. F. & Lapage, S. P. (1974). Effect of carbohydrates and related compounds on the long-term preservation of freeze-dried bacteria. Cryobiology 11, 73-9.

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Kirsop and J. J. S. Snell, pp. 23-33. London: Academic Press. Sourek, J. (1974). Long-term preservation by freeze-drying of pathogenic bacteria of the Czechoslovak National Collection of Type Cultures. International Journal of Systematic Bacteriology 24, 358-65.

Stamp, L. (1947). The preservation of bacteria by drying. Journal of General Microbiology 1, 251-65.

5 Identification L. R. HILL, K. A. MALIK and K. KOMAGATA

5.1

Introduction

Bacteria are ubiquitous and although the most comprehensive compendium, Bergey's Manual, lists only two to three thousand named species, the diversity of habitats means that no single identification scheme can be devised for the whole spectrum. With bacteria, the species definition itself is pragmatic, since a species is that which, for practical purposes, we find useful to consider as a species. A listing of only two to three thousand species names - a small number compared with, say, fungi, or insects - perhaps tells us more about the restraint of bacterial taxonomists rather than about bacterial diversity. It is certain that a unit currently considered to be 'species' in one of those parts of the total bacterial spectrum that has come under much study (e.g. pathogens of the human gut; antibiotic producing soil organisms) does not correspond in taxonomic rank with a 'species' in a littlestudied part of the spectrum. Identification to species level, however broadly or narrowly defined, is a major part of bacteriological practical work. However, it may be important to know not only to what species the organisms belong, but whether two or more isolates are, in fact, the same strain. For example, from multiple isolates in a hospital, can it be deduced whether a particular strain is spreading? Either way, all identifications have certain elements in common: examination of the current culture to determine as definitively as possible its characteristics, assessment of such data and comparison with the known characters of defined taxa, and finally, allocation of the unknown to a defined taxon or, if that is not possible, a tentative conclusion drawn that the unknown may belong to a new taxon and requires a fuller taxonomic study. 81

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5.1.1

Identification and taxonomy The difference between identification and 'full taxonomic study' is one of degree, the latter comprising three steps: classification, nomenclature, identification. For classification, a set of organisms is ordered into groups (taxa) on the basis of shared or common properties. The wider the range of properties determined, the more robust the classification will become in the sense that it is less likely to need future substantial revision as new information about the same organisms becomes available later. The shared properties become the definitions of the taxa; there are no formal rules as to how taxa should be defined. Nomenclature is the creation of names for defined taxa, and this is done in accordance with formal rules governed by international consensus through the Bacteriological Code of Nomenclature (analogous to the Botanical and the Zoological Codes). Nomenclature rules are very necessary to prevent the same name being given to different taxa, or different names to the same taxon, and to establish which of competing synonyms, when these occur, is deemed 'correct'. Identification, on the other hand, is allocation of new isolates to previously defined and named taxa and is usually carried out within constraints of time, practicability, and differing degrees of necessity, and is thus often based on fewer characters than would be used in a classification study. Knowledge about bacteria is continually increasing, and consequently ideas of how best to classify them necessarily change and evolve, sometimes with nomenclatural consequences. It must be remembered that identification is always carried out within the context of classifications, which themselves are changing. 5.1.2

Traditional and new methods For general purposes, and deriving from the traditional methods of examining bacteria, most current classifications (and, in turn, identifications) are based on observable macro- and micro-morphology, physiology, and ranges of biochemical reactions (or reactions to chemicals) effected by the organisms under a variety of conditions. This is sometimes called 'phenotypic classification' and is applicable over the whole spectrum of bacterial species. There concurrently exist various chemotaxonomic methods of phenotypic classification, which can yield a chemical fingerprint, or 'profile', of a bacterium. Identification can, in theory, be carried out exclusively with fingerprinting the unknown and matching with libraries of fingerprints previously

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determined for known and named strains: examination of fatty adds, cell walls (or particular components of these), total or selected proteins, are examples. Techniques of molecular biology further permit classification based on the genome: DNA base compositions, in vitro hybridisation between strands of DNA from different strains or endonuclease digest patterns are examples of such techniques. Differences and similarities between organisms observed at the phenotypic level can be more completely estimated by studying the genome. Chemotaxonomy and genomic study are rapidly expanding fields, but inevitably as yet restricted to those organisms which, for one reason or another, are important enough to justify the much greater effort required than is the case for traditional 'phenotypic classification'. However, some of the newer methods allow developments for identification methods that, ultimately, may be quicker and possibly cheaper, than traditional methods. For example, a specific DNA-probe can be made to detect rapidly a particular species; such a probe would be useful for field situations where the presence of the species would be significant, but where in its absence the identity of other bacteria present is not important. There are also many special identification systems, often important at subspecies level, such as the identification of serotypes within a species. In such situations identification may consist of determining solely these special properties. 5.1.3

Conventional identification

Identification traditionally consists of determining the morphology, physiology, biochemical reactions, resistances and sensitivities of the unknown organism. How many such characteristics are needed can never be stated a priori with certainty. Much will depend on whether circumstances, other than purely academic interest, require identification to genus, species, or subspecies level; much will also depend upon whether results obtained suggest a species in a poorly studied group. In this case, one may soon exhaust available tests and any uncertainty as to the identification will be more a reflection on the poor state of classification than upon inadequacies of the identification process. With most bacterial species definitions (in phenotypic terms), the possession or absence of many of the properties is rarely 100% constant over all studied strains of a particular species. Thus, identification keys

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(comparable with, say, botanical flora) have never been very successful with bacteria, or are so exceedingly complex and cumbersome so as to allow one and the same species to be reached by several different pathways through the key. Moreover, since most bacterial characteristics do not come from simple macro- or microscopic observation, but rather from growth experiments yielding chemical reactions, it is not usually possible to determine characters one at a time, as keys dictate, but rather in sets. There are two principal approaches to modern bacterial phenotypic identifications which both have their roots in medical bacteriology: probabilistic identification and identification kits. 5.1.4

Probabilistic identification Probabilistic identification uses tables, or matrices, of taxa and tests, and results are compiled not as simply positive or negative (present or absent), but rather as a statistical probability of being positive (present). Thus a taxon that has always been recorded as positive for a certain test will be scored with the highest probability (usually 0.99); and another taxon never found positive, with the lowest (usually 0.01). Within many taxa and many tests intermediate values will be found too. The test results for the unknown will necessarily be 1.0 or 0 (present or absent). These are matched against the record for each taxon separately in the matrix; corresponding probabilities are multiplied together, and one taxon will appear more probable than the others. Acceptance criteria have to be established, which in medical bacteriology may have to be rigorous. Databases and data processing of identifications can, and are, carried out on computers, and indeed such methods are sometimes called 'computer identification' or, better, 'computer-assisted identification'. Effectiveness of these methods depends on the adequacy of the matrix in terms of numbers of taxa and of tests and accuracy of the statistical probabilities entered in it. These probabilities can be amended and made more accurate as more and more strains are identified and new data added to the matrix. 5.1.5

Identification kits Identification kits partly derive from the above computerassisted, probabilistic methods. Evidently when devising classifications, it is pertinent to use as much information as practicable, and as wide a selection as possible of different properties of the organisms.

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Once taxa have been defined, then identification of unknown isolates becomes possible on fewer tests, providing these are in fact the defining tests. Over a range of taxa, the actual tests comprising definitions will not always be identical sets. A balance, therefore, has to be obtained between carrying out few tests yet with adequate coverage of the taxa to which the unknown is likely to belong. Good selection of reduced sets of tests for identification purposes can best be made by exploiting detailed information that comes from probabilistic identification systems. Once having made as good a selection as possible, then the basis for easy-to-use standard kits has been laid. There are now many competing commercially produced kits, which are miniaturised conventional tests, or sometimes tests for preformed enzymes. To stay small in numbers of tests, a kit usually has to be devised with a pre-defined major group of bacteria in mind, thus presupposing from either a few tests or from the isolation source, what kind of bacterium is likely to be found. Therefore kits developed for enterobacteriaceae differ from those for streptococci. It can be expected that the advantages of such kits are greater standardisation of the tests themselves - thus improving reproducibility, compactness, saving of labour, and acceptable shelf-lives. 5.1.6

Special phenotypic identification

Particularly within a species ('infrasubspecific division' of a species), it is sometimes necessary to identify whether an isolate is indistinguishable from another, or whether cultures from a whole series of isolations can be reasonably called 'the same strain'. With the development of immunology in the early years of this century, it was soon discovered that the antigenicity of bacteria can be very complex; certain antigens are common to different strains of the same species (and thus permit development work with vaccines), others are not in common. By determination of different antigens it became possible to define different serotypes within a species. These have since evolved to highly sophisticated, reliable and reproducible systems with certain organisms (e.g. Salmonella), and are of great practical utility in epidemiological studies of outbreaks, tracing original sources of these and so on. However, classical serological classification, and identification based upon it, suffer two principal defects: first, not all species have permitted the same degree of sophistication and success as has occurred with, say Salmonella (indeed, some species seem quite refractory to this

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approach); secondly, even when a serotyping system is developed, it is never fully exhaustive since there will always be a percentage of strains that cannot be typed. Such strains are not necessarily defective in their antigenicity, but rather are of a type that is not common, and no antiserum has been prepared. Recent developments in the more refined specific monoclonal antibody techniques may now have provided a new lease of life to serology. It is perfectly possible to make monoclonals strain-specific or, by selecting genomic parts that may be common to many strains of a given species, species-specific. In this same category of specialised identification is bacteriophage typing. Different strains of a given species may be more, or less, sensitive to particular phages, or resistant to others. Again, typing schemes based on sets of phages have been developed for certain species. 5.2

Practical aspects of identification

5.2.1

General considerations

Cultures to be identified should be pure cultures and should be stored adequately for future study and comparison. As a first step, microscopic (simple, phase contrast) examination of the strains should be performed for a purity check, detection of motility, cell shape and other morphological characters; Gram reaction, flagella number and position, and acid-fastness should be determined. Where facilities are available electron microscopy may prove helpful. During morphological examination, consideration should be given to the age of the culture, the medium used, growth conditions, cyst and spore formation, pleomorphism and coccoid body formation, all of which can affect observations. For characterisation, simple physiological and biochemical tests as well as highly specialised techniques such as serology, bacteriophage typing, genetic analysis, DNA-DNA homology, and ribosomal sequencing should be performed. However, depending upon the available facilities, it is important that the number of tests applied to identify a bacterium be kept to a minimum and that these should be as simple as is consistent with accuracy. From hundreds of characterisation tests available, a test pattern which will quickly and simply identify the organisms under study should be selected. The tests which best distinguish one group of organisms from another can be listed and a chart describing minimal characters for identification can be prepared. It is advisable to prepare one's own chart of 'Minimal characters' for

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the identification of a particular bacterium. Such charts are also available in various laboratories where identification is done routinely or is offered as a service. At the Deutsche Sammlung von Mikroorganismen und Zellkulruren (DSM), charts on minimal characters for the identification of Azospirillum, Bacillus, Clostridium, Pseudomonadaceae, and of some physiological groups such as hydrogen oxidising bacteria or nitrogen-fixing bacteria have been in long use. Knowledge of the source of the strain and the conditions under which it was originally isolated is often helpful in delimiting the genera to which the organisms may belong. Such information, together with morphology and Gram staining reaction, usually enables the investigator to narrow his search to a small group of genera. Based on the information of initial isolation procedures, relatively difficult physiological division of bacteria into groups of chemolithotrophic autotrophs, photosynthetic bacteria and chemoheterotrophic bacteria can be performed. Requirement for oxygen and utilisation of carbohydrates oxidatively or fermentatively proves helpful in bacterial subdivision into major groups (aerobic, anaerobic, facultative anaerobic, oxidative, fermentative, and so on). After preliminary identification, cultures of reference and type strains of related species should be acquired for comparison. 5.2.2

Purity of cultures Bacteria isolated directly from animals, plants, soil or the general environment are rarely obtained in pure culture. Thus the first step in identification is to obtain each organism in pure culture. The techniques used vary with the organism being studied. Selective or inhibitory media are often used for isolation and single colonies of desired bacteria are isolated ('picked') from mixed populations. Singlecolony selection from such plates does not assure purity, as non-growing viable contaminants may be picked along with the chosen bacteria. Selective media should be avoided in the purification process. 5.2.3

Standardised tests and identification schemes There are several simple biochemical and physiological tests which are frequently useful in identification of bacteria to the species level. These include: the presence of oxidase, catalase or urease, nitrate reduction, hydrogen sulphide production, acid or gas production from sugars, temperature range of growth, antibiotic sensitivity, nutritional requirements, ability to utilise specific substrates, metabolism of

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hydrogen, nitrogen and amino acid metabolism, production and hydrolysis of poly-B-hydroxybutyrate and finally some additional tests selected by screening the characteristics for the cluster of genera to which the bacterium has been assigned. However, for species which cannot be characterised by simple traditional tests, recourse to chemotaxonomic study may be necessary. A variety of convenient and rapid multitest systems (API System, Minitec System, Enterotube-Roche, R-B System, Patho-tech System, Auxotab, etc.) is commercially available along with the charts, tables, coding systems and characterisation profiles. No single system has yet been agreed for universal use with any given taxon. Thus, when making use of new identification systems or using a new identification scheme it is always advisable to include types of reference strains (whose identity is established) in comparative identification. Also it is essential to use identical or standard conditions for performing the tests upon which such schemes are based. 5.2.4

Evaluation of results Test results should be recorded at regular intervals and for some, such as colonial and cellular morphology, for up to perhaps 1-2 weeks. For well known species, comparison of final test results with published descriptions is the usual practice for reaching an identification. For little known species, however, a further evaluation may be necessary, through study of a known reference or type culture of the species with the tests carried out under the same conditions used for the unknown culture. 5.2.5

Assignment of new names It may arise, from time to time, that the culture under examination does not identify to any known species. To propose and name a new species, the rules of the International Code of Nomenclature of Bacteria must be followed, a type strain designated and deposited in one of the permanently established culture collections. The new species and its name becomes valid only when it is published in the International Journal of Systematic Bacteriology. It should be recognised, however, that the majority of isolates that are difficult to identify are atypical strains of existing species rather than new species.

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5.3

Chemotaxonomy A considerable volume of recent and current bacterial classification studies is based upon chemotaxonomic methods. Most of these methods require special apparatus and skills for their successful application. They can also be used as identification methods, either supplementing conventional phenotypic methods or, at least in theory, standing alone. Routine use of chemotaxonomic methods may well be possible only in specialised laboratories, but given the importance of these approaches to our fuller understanding of relationships between species, a culture collection may well be in such a specialised laboratory, especially if it has a research programme related to one or other aspect of taxonomy. Peptidoglycan is a characteristic cell wall polymer of Gram-negative and Gram-positive bacteria and is almost uniform in Gram-negative species. Variations in the primary structure of the peptidoglycans of various Gram-positive bacteria are of high taxonomic importance. Archaebacteria, having ether-linked lipids in their cell wall, can be differentiated from eubacteria possessing ester-linked lipids. Other lipids with chemotaxonomic potential are polar lipids, hopanoids, hydrocarbons and carotenoids. Fatty acid patterns may be characteristic for a particular taxon and a special category of these acids, 'mycolic acids', are only found in the taxa Bacterionema, Corynebacterium, Micropolyspora, Mycobacterium, Nocardia and Rhodococcus.

Cytochrome patterns too are a useful aid in bacterial classification. Lactic acid bacteria contain only cytochrome b, and cytochrome c is often absent from facultatively anaerobic Gram-positive bacteria. The genus Clostridium lacks cytochromes. Cytochrome d is characteristic of many Gram-negative bacteria. Chemotaxonomic methods applied to bacteria range from pyrolysis gas-liquid chromatography of whole cells, to extraction and comparison of defined subcellular fractions (e.g. fatty acids, quinones, components of cell walls, proteins), of the nucleic acids, of fragments of nucleic acids and, perhaps the ultimate level so far as identification applications are concerned, highly specific nucleic acid probes, even for particular genes. Chemotaxonomic methods yield qualitative and quantitative data, and an important feature of several, but not all, is that the particular class of chemical components can be subjected to one method or another of molecular separation, recordable as a quantitative chemical profile or 'fingerprint'. Using computers, these can be digitised and the

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fingerprints from the same organisms on different occasions can be compared to establish levels of reproducibility. Finally, comparisons can be made between different organisms. Databases of profiles can be made and an identification of an unknown isolate is then simply achieved by obtaining a profile and using a computer to compare it with the different profiles in the database to find the most similar, or identical, one. 5.3.1 Chromatography Pyrolysis gas-liquid chromatography. This is a method in which whole

cells are 'burned' in their entirety, the combustion products separated by gas-liquid chromatography (GLC) and the differing proportions of different chemicals recorded as a series of peaks and troughs. The method can be used in conjunction with mass spectrometry, by which the chemical nature of each of the peaks can be determined. Extraction, purification and separation by GLC of short-chain fatty acids has been used; volatile acids can be directly separated by GLC, non-volatile acids can be first converted to volatile eaters and then chromatographed. Isoprenoid quinones are very widely distributed among microorganisms, they are involved in electron transport and can be separated by thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC). Sugars and amino acids as cell wall components have been used for bacterial differentiation, again using chromatography methods. Electrophoresis of proteins. Proteins are components of all cells and any one bacterial cell may have as many as 2000 different proteins. The overall protein composition can be very different from one species to another. To differentiate cells in this way, either total cellular proteins or outer membrane proteins are extracted from cell lysates and stabilised with sodium dodecyl sulphate (SDS) and subjected to one- or two-dimensional separations either by polyacrylamide gel electrophoresis (SDS-PAGE) or by SDS-PAGE in one dimension and isoelectric focusing in the other dimension. With one-dimensional SDSPAGE, it is usual to obtain fifty to sixty discrete protein bands, visualised by staining or, if a radiolabel has been incorporated into the proteins prior to extraction, by autoradiography. The bands may comprise more than one protein (since with two-dimensional separations some 1500-2000 separate spots are obtained), but a 50-60 band profile has sufficient complexity to permit recognition of profiles typical of

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particular species, and minor differences between profiles from different strains of the same species. For practical classification and identification work, one-dimensional separation is adequate; twodimensional separations are generally of a more academic interest, or may be used to detect the presence or absence of a particular protein. One-dimensional separations enable several extracts (different strains, marker or reference strains, molecular weight markers) to run as separate tracks on a single gel. Visual inspection is often used, or strains can be scanned by a densitometer which, in turn, can be interfaced with a computer; this enables statistical comparisons between tracks (the most widely used algorithm being the correlation coefficient) and, importantly, the storage of a database of reference profiles.

5.4 5.4.1

Nucleic acids DNA base compositions The similarity or dissimilarity between two organisms will be found in the base sequence which codes for the proteins. For a given organism, the value of the percentage of guanine and cytosine bases of the DNA (% G+C) is constant. As the overall base composition in bacteria varies from about 25% G+C to 75% G+C, bacteria differing in DNA base composition by about 10% G+C or more, will have only a very small proportion of segments of DNA of the same base composition, or none at all. Organisms with the same base composition may still, however, be unrelated, since one and the same base sequence may have the same overall base composition, but an entirely different sequence made up of the same proportions of the four different bases. It follows then that % G+C can be used taxonomically only in a negative sense, indicating only whether compared bacteria are different. In identification, determination of % G+C can be used to help indicate in which general part of the bacterial spectrum the unknown organism lies, or to exclude tentative identifications. Determination of % G+C is usually indirect, by thermal denaturation or buoyant density measurements. 5.4.2

Base sequence similarities In vitro molecular hybridisation of DNA:DNA or DNA:RNA indicates quantitatively degrees of matching in base sequences between nucleic acids from different bacteria. The procedures for carrying out such molecular hybridisations do not lend themselves to

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routine identification work, despite many attempts to devise batch methods of processing simultaneously multiple samples of nucleic acid. Nevertheless, the results from nucleic acid hybridisation experiments, carried out in a taxonomic framework, are often taken by taxonomists as the ultimate arbiter to make taxonomic revisions or define limitations to species. DNA homology values are average measurements of similarity in which the entire genome of one organism is compared with that of another, whereas RNA homology values are specific for each type of RNA (messenger RNA, ribosomal RNA and transfer RNA). Many procedures such as direct binding method, binding in agarose gel, binding to nitrocellulose and free solution reassociation methods have been developed for homology determinations. 5.4.3

DNA fingerprinting Differences and similarities in base sequences can be revealed indirectly through analysis of fragments of DNA separated by agarose electrophoresis after the DNA has been digested by specific restriction endonucleases. There are many such enzymes that cleave DNA only at specific points defined by four to six bases to one side of the cleavage site. Thus, digestion with a particular endonuclease of DNA from one organism will give rise to a characteristic number of fragments (of differing sizes), dependent on the number of recognition sites. The fragments are then separated electrophoretically and a characteristic fragment-profile, or DNA fingerprint, will result. Treating DNA from other organisms in the same way, with the same enzyme, will yield comparable fingerprints. As with protein-profiling, analysis of such fingerprints can be simply visual or automated; again, the use of computers permits the creation of databases and then lends itself to molecular identification. 5.4.4

DNA probes By exploiting the specificities of endonucleases and the sharp separation of fragments, it is possible to make DNA-probes. These are in vitro synthesised DNA from selected fragments, which can be at a generic level (e.g. fragments corresponding to RNA genes, which are evolutionarily conserved, and thus show similarities over wider ranges of different genera and species), at species level or yet more specifically at a subspecies level. The specificity can be even more fine-tuned, to

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detect, say, one particular gene, for example a gene producing a particular toxin. In this latter case, the same toxin may occur in different species, so a highly specific DNA probe of this type cannot be used for identification in the usual sense; they serve to identify particular traits that for one reason or another are of interest. For medically important bacteria, the rapid identification of particular pathogens can be facilitated by DNA-probes, some of which are already commercially produced in relatively easy-to-use kit forms. Bacterial identification by these means is limited, first, by the numbers and specificities of probes currently available, and second, because a positive result gives an identification, while a negative one yields no information other than exclusion of the organism for which the probe was designed. 5.5

Selected references for identification Buchanan, R. E. & Gibbons, N. E. (1974). Bergey's Manual of Determinative Bacteriology, 8th edn. Baltimore, MD: Williams and Wilkins. Cowan, S. T. (1974). Cowan and Steel's Manual for the Identification of Medical Bacteria. Cambridge: Cambridge University Press. Feltham, R. K. A., Wood, P. A. & Sneath, P. H. A. (1984). A generalpurpose system for characterizing medically important bacteria to genus level. /. Appl. Bacteriol. 57, 279-90. Goodfellow, M. & Board, R. G. (ed.) (1980). Microbiological classification and identification, Society for Applied Bacteriology Symposium Series No. 8. London: Academic Press. Johnson, J. L. (1981). Genetic characterization. In Manual of Methods for General Bacteriology, ed. Gerhardt et al., pp. 450-72. Washington, DC: American Society for Microbiology. Krieg, N. R. & Holt, J. G. (1984). Bergey's Manual of Systematic Bacteriology, Vol. 1. Baltimore, MD: Williams and Wilkins. Mandel, M., Igambi, L., Bergendahl, J., Dodson, M. L. & Scheltgen, E. (1970). Correlation of melting temperature and cesium chloride buoyant density of bacterial deoxyribonucleic acid. /. Bacteriol. 101, 333-8. Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. /. Mol. Biol. 5, 109-18. Mitruka, B. J. (1976). Methods of Detection and Identification of Bacteria. Cleveland, Ohio: CRC Press. Skerman, V. B. D. (1967). A guide to the Identification of the Genera of Bacteria, 2nd edn. Baltimore, MD: Williams and Wilkins. Sneath, P. H. A., Mair, S. N., Sharpe, M. E. & Holt, J. G. (1986). Bergey's Manual of Systematic Bacteriology, Vol. 2. Baltimore, MD: Williams and Wilkins.

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H. G. Triiper, A. Balows and H. G. Schlegel. pp. 176-93. Berlin: Springer-Verlag. Willcox, W. R., Lapage, S. P. & Holmes, B. (1980). A review of numerical methods in bacterial identification. Antonie van Leeuwenhoek ]. Microbiol. Serol. 46, 233-99.

6 Patent protection for biotechnological inventions I. J. BOUSFIELD

6.1

Introduction

This chapter is intended to give the reader who is unfamiliar with patents an introduction to the patent system as it applies to biotechnology, and a general guide to the procedures and pitfalls involved in obtaining patent protection for biotechnological inventions. For a detailed discussion of the whole subject of patents in biotechnology and a review of the variety of national patent systems the reader is referred to the excellent texts by Crespi (1982), Crespi & Straus (1985) and Straus (1985). It is not possible here to provide a stepby-step guide to getting a patent in every country in the world, for, despite an overall similarity, variations between different national patent laws are manifold, and professional help is necessary to guide even the experienced inventor through their complexities. The present account does no more than skim the surface of what is a complex and often fascinating subject; for this reason a short list of selected publications which illustrate in more detail many of the points raised here is given in Section 6.6, Further reading. 6.2

Basis of the patent system

6.2.1

Principles

The principle (if not the practice) of the patent system is straightforward: the inventor of a new product or process publicly discloses the details of his invention and in return he is granted for a limited period a legally enforceable right to exclude others from exploiting it. In this way the inventor's ingenuity is acknowledged and rewarded, while at the same time further technical progress is encouraged by the public dissemination of information about the invention. 95

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6.2.2

Criteria for patentability

To qualify for patent protection, an invention must meet the following major criteria. Novelty. The invention must be new. Most countries apply the test of 'absolute novelty', which means that if prior knowledge of it exists anywhere in the world (not merely in the country where patent protection is sought) then the invention belongs to the state of the art ('prior art') and is not patentable. The prior art is held in these countries to include anything the inventor himself may have said or written about his invention. Exceptions to this rule of absolute novelty are the patent systems of the USA and Canada, where publications by the inventor made not more than one (USA) or two (Canada) years before a patent application is filed in that country do not destroy novelty. 'Grace periods' of six months are also allowed by Australia, New Zealand and Japan, but only in respect of certain kinds of publications made by the inventor, for example at certain scientific meetings. Under nearly all patent systems, publications made after the date that the patent application is filed (the 'priority date') do not jeopardise protection for that particular subject matter in that particular application. It must be remembered, however, that they will form part of the prior art against which any future applications will be assessed. Inventiveness. The invention must show evidence of an inventive step, that is it must not be 'obvious' from the state of the art to anyone 'skilled in the art'. In simple terms this means that the average expert in the field under consideration could not reasonably have predicted the invention as an obvious or logical outcome of what he already knew. Utility (industrial applicability). The invention must have a practical use, which in the USA means exactly that, but in nearly all other patent systems means that it must also be capable of industrial application. Most countries, however, hold that medical methods for the direct treatment of the human or animal body are not susceptible to industrial application and are therefore not patentable. The major exception to this is again the USA. Disclosure. The details of the invention must be disclosed by means of a patent specification (see Section 6.5.2 below) so that the invention is

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described in sufficient detail to allow a skilled person to reproduce it. This is normally done by means of a written description supplemented where necessary by drawings. However, in one major category of biotechnological inventions - those involving the use of new microorganisms and certain other novel living materials - a written description is not usually considered sufficient for the purposes of disclosure. In such cases it is argued that no matter how carefully the description may be worded, if the microorganism itself is not available, the invention cannot be reproduced. Therefore, many countries require the deposit of new microorganisms in a recognised culture collection to ensure their public availability. This unique aspect of biotechnological patent procedure is dealt with in some detail later in this chapter. Exclusions for patentability. As well as meeting the criteria listed above, an invention must not be of a kind which is excluded from patentability by its very nature. Exclusions relating specifically to biotechnological inventions are discussed later, but in general terms patents cannot be obtained for mere discoveries, theories, computer programs, literary works, musical compositions, aesthetic creations and illegal or offensive devices. 6.3

Kinds of biotechnological inventions There are four main kinds of biotechnological invention: products, compositions, processes, and use or methods of use (Crespi, 1982). 6.3.1

Products

These inventions are exactly what the word suggests - tangible new materials or entities. They include organisms themselves (e.g. bacteria, fungi), parts of organisms (e.g. cell lines), substances produced by either of these (e.g. enzymes, antibiotics), and substances obtained by or employed in recombinant DNA techniques (e.g. plasmids, DNA molecules). Product inventions can be the subject of two broad kinds of patent claim: the 'product per se', where patent protection is sought for the product itself, regardless of the method of manufacture, and the more limited 'product-by-process', where protection is sought for the product obtained by a particular process.

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6.3.2

Compositions These inventions are mixtures of substances or organisms, the individual components of which may already be known, but which in combination can be shown to display a new property or exert a new effect. 6.3.3

Processes These inventions are methods for the manufacture of products, and include bioconversions, fermentations, and methods of isolation, purification or cultivation. Some process inventions are genuinely new methods for obtaining novel or known products, but others are known methods applied in new situations or used in the manufacture of novel products. 6.3.4

Use and methods of use Methods of use include processing or treating materials (e.g. industrial raw materials or agricultural products), non-medical treatments of humans or animals, 'off the body' medical methods (e.g. a method of diagnosis carried out on a sample taken from a patient), methods of testing (e.g. quality control) and in a few countries, notably the USA, medical treatment of humans and animals. Also, the new medical use of a substance previously unknown to have that use is protectable in European Patent Convention (EPC) countries. 6.4 6.4.1

Patentability of biotechnological inventions Inventions involving new plants and animals Plant varieties. By far the most common form of legal protection for new plant varieties is the plant variety right (although in the USA special 'plant patents' are available for asexually propagated plants). Several countries are now party to the International Convention for the Protection of New Varieties of Plants (UPOV) which aims to harmonise national practices as far as possible (International Convention, 1978). In these countries, plants that are protected by plant variety rights are usually specifically excluded from patentability. Plant variety rights in general are intended to allow the commercial plant breeder a monopoly on the production of propagating material for the purposes of commercial marketing, its offer for sale and its marketing. Plant variety rights are easier to obtain than is patent protection as there is no requirement for inventiveness or for reproducible disclosure, but they are more limited in scope in that neither the plant itself nor consum-

Patent protection for biotechnological inventions ables produced from it (e.g. fruit for eating, grain for milling) are protected. Plant variety rights were introduced essentially to cover varieties developed by traditional breeding methods and it is such varieties that are excluded from patentability in many countries. Thus Article 53(b) of the EPC, to which 13 countries belong (Table 6.1) states the following: European patents shall not be granted in respect of . . . plant or animal varieties or essentially biological processes for the production of plants or animals; this provision does not apply to microbiological processes or the products thereof. The same exclusion is found where the national laws of individual European countries have been harmonised with the EPC and is also contained in the laws of several other countries, e.g. the German Democratic Republic, Mexico, Sri Lanka, Thailand and Yugoslavia. Plant and animal varieties, but not essentially biological processes, are also excluded from patentability in China. The exclusion of plant varieties from patent protection is a contentious issue. Straus (1985) has pointed out that current systems for the protection of plant varieties were introduced when plant breeding methods did not permit the breeder to fulfil the normal criteria of patentability. However, the advent of new technologies, particularly genetic manipulation techniques, for the production of new plant varieties has meant that these requirements can now be met. Therefore, there seems to be a good argument in favour of allowing the developer of such varieties the right to choose between protection under the patent system or through plant variety rights (Beier et ah, 1985; Straus, 1985).

Table 6.1. Countries party to the European Patent Convention at 1 January 1987

Austria Belgium France Germany (Federal Republic) Greece Italy Liechtenstein

Luxembourg Netherlands Spain Sweden Switzerland United Kingdom

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Animal varieties. Although a special form of protection for new animal varieties (which are not, however, regarded as inventions) is available in the Soviet Union, there is in general no special system of legislation for their protection. Most of the countries that exclude plant varieties from patentability also exclude animal varieties, and in those countries that do not, the position is not altogether clear. However, recent court decisions in the USA (in re Diamond & Chakrabarty, 1980) and Canada (in re Abitibi, 1982) suggest that animals may be patentable provided that the requirements for enabling disclosure, that is repeatability, are met (Straus 1985). Processes for the production of animals and plants. Many patent laws,

including those of the EPC, specifically deny patent protection for 'essentially biological processes' for the production of plants or animals; microbiological processes, however, are not included in this provision. This terminology may not be entirely clear and perhaps needs some explanation. Stated simply, an essentially biological process is considered to be one in which the result is achieved with a minimum of human technical intervention. The example given by the European Patent Office (EPO) in its guidelines to examiners is a method for selectively breeding horses in which human intervention is limited to bringing together animals having particular characteristics. On the other hand, a process for treating a plant to promote or suppress its growth, e.g. a method of pruning or of applying stimulatory or inhibitory substances, would not be considered to be essentially biological, since although a biological process is involved, the essence of the invention is technical. Given this definition of 'essentially biological' and the exemption of microbiological processes from it, the normal criteria for patentability can be applied to methods for producing plants by, for instance, genetic manipulation involving the use of vectors in microbial hosts, or by somatic cell hybridisation. However, matters are less certain under the EPC in respect of some processes for the production of new animal varieties, even though such processes may meet the test of not being essentially biological. This is because a further exclusion from patentability is found in Article 52(4) of the EPC, which states the following: Methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body shall not be regarded as inventions which are susceptible of industrial application . . . [See also Utility in Section 6.2.2 above.]

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Straus (1985) has expressed the fear that some present and future approaches to animal breeding, such as techniques of embryo transfer, could be denied patent protection by this provision. In support of his argument he cites a recent decision by the UK Comptroller of Patents, in which an application involving just such a technique was rejected as being a method of treatment by surgery. In contrast to the European system, the patent laws of the USA, Japan and China do not exclude essentially biological processes. Furthermore, the US laws do not exclude methods for treating animals (or humans); therefore the problems presented by the European system in respect of patenting processes involved in animal breeding do not exist in the USA. Tissue cultures. Animal cell lines and plant tissue cultures (and in Japan, seeds) are generally considered to be in the same category as microorganisms for patent purposes. Thus they are subject to the provisions applied to microbiological inventions as discussed below. As regards plant cells, however, the US Patent Office makes a distinction between undifferentiated cell lines used, for instance, to produce a particular substance, and cells which are capable of differentiation and which are used simply to reproduce the whole plant. 6.4.2

Inventions involving microorganisms

Applied microbiology in its broadest sense is a major facet of modern biotechnology, and any discussion of patents in biotechnology inevitably must focus on the peculiar problems posed by microbiological inventions. In fact so great has been the attention given to these problems in patent circles that the patent legislation of an increasing number of countries contains specific provisions for inventions involving the use of microorganisms, and one international convention (the Budapest Treaty; see below) deals entirely with microorganisms (Budapest Treaty and Regulations, 1981). It should be said that the term 'microorganism' is used in patent circles in a very loose sense and encompasses entities that strictly speaking are not microorganisms, e.g. cell lines and plasmids. Indeed, the word is intentionally not defined in the Budapest Treaty so as to avoid undue constraints being imposed upon the application of the Treaty, and in the words of the World Intellectual Property Organization (WIPO) commentary on the draft Treaty (WIPO, 1980), it 'need not correspond to usage in some scientific circles'. Unfortunately, the use of such inexact terminology has led to uncertainty in some quarters as

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to what is or is not a microorganism. Because of this, the present author (acting on behalf of the World Federation for Culture Collections (WFCC) Patents Committee) has proposed to WIPO that the expression 'living material' be used instead of the word microorganism, particularly in regard to the Budapest Treaty. The word 'living' was defined as 'that material which under appropriate conditions is able to replicate itself, or which at least possesses the functional genes necessary to direct its own replication'. This definition has two advantages: first, it avoids insoluble philosophical arguments about where chemical reactivity ends and life begins, and second, it excludes nonliving biological materials such as enzymes. In the present chapter, although the word microorganism is used for ease of reference, it should be taken to mean living material as defined above. Microbiological inventions may be found in all of the categories of biotechnological invention outlined in Section 6.3 above. In general, microbiological processes and the (inanimate) products obtained by them can be considered analogous to chemical processes and products, and obtaining patent protection for them in most countries is nowadays fairly straightforward, provided that the basic criteria for patentability are fulfilled. Less straightforward, however, is the patenting of microorganisms as products either per se or as products-byprocess. It is these inventions above all others that demonstrate the difficulties of determining the borderline between 'discoveries' and 'inventions', of what is 'new' and what is not, and of ensuring sufficiency of disclosure. Naturally occurring microorganisms. A previously unknown naturally occurring microorganism that is left in its natural state is universally regarded as a discovery and is unpatentable. However, the degree of human intervention considered necessary to turn such a discovery into a patentable invention (assuming it has a practical use) varies between different countries. The extent of this variation was demonstrated by the official replies to a questionnaire on patent protection in biotechnology distributed to governments in 1982 by the Organization for Economic Cooperation and Development (OECD). These responses were reviewed in detail by Crespi (1985). In those countries that do permit naturally occurring organisms to be patented, isolation and purification of the organism are general prerequisites, after which various constraints are applied, mainly relating to novelty and unexpected properties. Thus, for example, the UK and the EPO require an

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organism to be 'new' in the sense of being hitherto unknown, whereas in Canada a new organism is one that does not already exist in nature (in this connection, Crespi (1985) has commented pointedly on the illogicality of equating 'unknown' with 'not previously existing'); the Federal Republic of Germany requires that 'certain changes occur during isolation so that the isolated microorganism is not identical with that occurring in nature'; Denmark requires naturally occurring organisms to have unforeseen properties. The USA permits the patenting of naturally occurring organisms as 'biologically pure cultures'. Non-naturally

occurring microorganisms. After the much-publicised

Chakrabarty case in the USA in 1980, in which a genetically manipulated strain of Pseudomonas was held not to be a product of nature but a human invention patentable per se, there are unlikely to be any unusual problems in obtaining patent protection for 'artificial' microorganisms (bacterial recombinants, hybridomas, etc.), other than in countries which do not permit the patenting of any kinds of microorganism. Sufficiency of disclosure. As already mentioned (Disclosure, in Section 6.2.2 above), one of the fundamental requirements of the patent system is that the details of an invention must be disclosed in a manner sufficient to allow a skilled person to reproduce the invention. Microbiological inventions present particular problems of disclosure in that more often than not repeatability cannot be ensured by means of a written description alone. In the case of an organism isolated from soil, for instance, and perhaps 'improved' by mutation and further selection, it would be virtually impossible to describe the strain and its selection sufficiently to guarantee another person obtaining the same strain from soil himself. In such a case, the strain itself forms an essential part of the disclosure. In view of this an increasing number of countries require a 'new' microorganism (i.e. one not already generally available to the public) to be deposited in a recognised culture collection whence it can subsequently be made available at some stage in the patent procedure. As a general principle, an invention should be reproducible from its description at the time that the patent application is filed. In the case of an invention involving a new organism, therefore, most patent offices require a culture of the organism to be deposited not later than the filing date of the application (or the priority date if priority is claimed from an earlier application - see Section

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6.5.3 below). Exactly when a strain becomes available varies according to the patent laws of different countries, and is a much debated question dealt with in more detail below (Release of samples). Since an invention must also be reproducible throughout the life of the patent, a microorganism deposited for patent purposes must remain available for at least this length of time. Most countries provide for a considerable safety margin in this respect, and availability for at least 30 years is a common requirement. The Budapest Treaty. In order to obviate the need for inventors to deposit their organism in a culture collection in every country in which they intend to seek patent protection, the 'Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure' was concluded in 1977 and came into force towards the end of 1980 (Budapest Treaty and Regulations, 1981). Under the Budapest Treaty certain culture collections are recognised as 'International Depositary Authorities' (IDAs), and a single deposit made in any one of them is acceptable by each country party to the Treaty as meeting the deposit requirements of its own national laws. Any culture collection can become an IDA provided that it has been formally nominated by a contracting state, which must also provide assurances that the collection can comply with the requirements of the Treaty. At 30 November 1989 there were 20 IDAs and they and the kinds of organisms they accept are listed in Table 6.5 below. The Budapest Treaty provides an internationally uniform system of deposit and lays down the procedures which depositor and depository must follow (see Section 6.5.5 below), the duration of deposit (at least 30 years or 5 years after the most recent request for a culture, whichever is later), and the mechanisms for the release of samples. The Treaty does not, however, concern itself with the timing of deposit nor, in the main, of release; these are determined by the relevant national laws. Likewise, the recipients of samples (other than patent offices and people with the depositor's authorisation) are referred to merely as 'parties legally entitled': exactly who such parties are and under what conditions they may obtain samples are again determined by national law. Twenty one states and the European Patent Office (EPO) are now party to the Budapest Treaty and are listed in Table 6.2. National deposit requirements. The requirements of various countries for the deposit and release of microorganisms for patent purposes are

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Table 6.2. Countries party to the Budapest Treaty at 31 July 1987

Australia Austria Belgium Bulgaria Denmark Finland France German Democratic Republic Federal Republic of Germany Italy Hungary Japan

Liechtenstein Netherlands Norway Philippines South Korea Spain Sweden Switzerland UK USA USSR European Patent Office (EPO)fl

a

The EPO is not, strictly speaking, a party to the Treaty, since it is not a country but an intergovernmental organisation. Article 9 of the Treaty provides for such organisations to file a declaration stating that they accept the obligations and provisions of the Treaty. The EPO has filed such a declaration. summarised in Table 6.3. Deposit is a statutory requirement under Rule 28 of the EPC and under the national laws of several of its member countries. In those EPC countries not having a statutory provision under their national law, deposit is such an established requirement of patent offices that it amounts to the same thing for all practical purposes. Most of these countries follow EPC practice in requiring the deposit to be made by the filing or priority date. An exception to this is the Netherlands, where deposit is required before the second publication (see next section) of the patent application. Most other European countries do not have specific requirements as yet, but nevertheless advise that deposits should be made, usually along the lines of the EPC. In many countries outside Europe, deposit is an established or recommended practice, and some patent offices (in Japan, USA, USSR, for example) have specific requirements for deposit. In almost all cases deposit must be made by the filing or priority date. In the USA, however, as a consequence of a recent court decision (in re Lundak, 1985), deposit may in certain circumstances be made after filing but before the issuance of a US patent. All countries party to the Budapest Treaty (Table 6.2) must recognise a deposit made in an IDA but not all require deposits to be made in ID As. Thus, for example, France, Germany, Switzerland, the UK, the USA and the EPC will recognise other culture collections that can

Table 6.3. National requirements (mandatory or recommended) for deposit and release of microorganisms*

Country

Deposit by Earliest release

Earliest general availability"

Australia Austria Belgium Bulgaria Canada Denmark Finland France Germany

F/P F/P F/P F/P F/P F/P F/P F/P F/P

1st pub. 1st pub. grant grant 1st pub. 1st pub. 1st pub. 1st pub.

1st pub. grant grant grant grant grant grant 1st pub.

Hungary

F/P

1st pub.

1st pub.

F/P F/P

2nd pub.

2nd pub.

Liechtenstein Netherlands New Zealand Norway Portugal Spain Sweden Switzerland UK

2nd pub. F/P F/P F/P F/P F/P F/P F/P

2nd pub. 1st pub. 1st pub. 1st pub. ? 1st pub.

2nd pub. grant 1st pub. grant ? 1st pub.

USA6

F/P

grant

grant

F/P

1st pub.

grant

Italy Japan

F/P r/±

Restrictions on distribution and use of samples as for UK as for EPO as for UK none as for EPO as for EPO as for EPO until patent expires, sample must not be passed to 3rd parties or outside purview of German law sample must not be passed to 3rd party as for EPO sample must not be passed to third parties until patent expires and must be used only for research purposes as for Switzerland none as for EPO as for EPO sample must not be passed to 3rd parties sample must not be passed to 3rd parties until patent expires and must be used only for experimental purposes none

Minimum storage period (years) 30 30 life of patent 30 30 30 20 20 life of patent

life of patent 30 30 30 30 30

TTCCR \JOO1S.

EPO

if applicant chooses, available only to independent expert before grant; must not be passed to 3rd parties before patent expires and must be used only for experimental purposes

30

F/P, filing or priority date as applicable; pub., pubication of application; -, no provisions or provisions not known; ?, conflicting information from different sources. *Author should be contacted for information subsequent to publication of the first books in this series (Filamentous Fungi, Yeasts) in 1988. " General availability means sample publicly available at least in country where application has been filed. b The Lundak decision (1985) may mean that in certain cases deposit may be made later.

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comply with their particular requirements. Hungary accepts deposits made in collections on its own soil, but the only deposits it will recognise elsewhere are those made in IDAs. The Japanese patent office, however, will recognise deposits outside Japan only if they have been made under the Budapest Treaty or have been 'converted' to Budapest Treaty deposits (see 'Converted' deposits in Section 6.5.5 below), regardless of their previous public availability. It must be remembered that deposits made under the Budapest Treaty can only be made in ID As. Most countries not party to the Budapest Treaty accept deposits made in any internationally known culture collection which will comply with their requirements; in some cases the collection is required to furnish a declaration as to the permanence and availability of the deposit. Release of samples. Microorganisms deposited to comply with requirements for disclosure must become available to the public at some stage of the patenting procedure. Unlike a written description, however, the microorganism is the physical essence of the invention itself and because of this the exact conditions of release are a matter of great concern to patent practitioners. There are as yet no internationally uniform release conditions, but three main kinds of system operate at present. In the USA, patent applications are not published until the patent is granted, and a microorganism deposited for patent purposes need not be made available until then. From the date of grant, the organism must be publicly available without any restriction. The major advantage of this system to the inventor is that his microorganism does not have to be released until he has an enforceable right. Furthermore, if he is not granted a patent then his microorganism need never become available. Thus under the US system an inventor is never put in the position of having to allow access to his organism when he has no legal protection. In Japan and the Netherlands patent applications are published twice, first 18 months after the filing date or priority date and before the application has been examined (Section 6.5.4 below), and second (for the purposes of opposition of third parties) when the application has been accepted. A microorganism deposited in connection with the application must be made available at the date of second publication, i.e. once the patent office has decided to grant a patent. Thus under the

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Japanese and Dutch systems the inventor again has an enforceable right at the time he is required to make his organism available. In Japan he is afforded a further measure of protection in that recipients of cultures must not pass them on to third parties and must use them only for experimental purposes. This provision does not apply under Dutch law, however. A dual publication system is also operated by the EPC and by the countries party to it. However, in contrast to the Japanese requirements, a microorganism deposited in connection with an EPC application must be made available at the date of first publication, i.e. before any enforceable right exists. This practice reflects the prevailing philosophy of European patent authorities that the organism is regarded as an integral part of the disclosure and therefore should become available at the same time as the written description. Originally cultures had to be available to anyone requesting them, subject to the recipient giving certain undertakings of rather doubtful value, but in response to pressure from users of the system the appropriate rule (Rule 28) of the EPC was amended to provide more protection for the inventor. Rule 28(4) now permits the applicant to opt to restrict the availability of his organism at first publication to an independent expert acting on behalf of a third party. The expert, who is chosen by the requesting party from a list held by the EPO, is not permitted to pass cultures of the strain to anyone else. After second publication, the strain becomes generally available, but at this stage the inventor has an enforceable right. Although this so-called 'expert solution' applies in respect of applications filed with the EPO itself, it is at present part of the national law of only a minority of member countries of the EPC (France, Italy, Sweden). None, however, permits recipients of cultures to pass them on to third parties. Need for deposit. In principle most countries require deposit only when repeatability of the invention cannot be ensured without it. Thus, for example, it should not be necessary to deposit a new recombinant strain if the procedure for constructing the novel plasmid and transforming it into a host can be described in sufficient detail to allow an expert to produce the same recombinant for himself (given, of course, that the original vector and host are already generally available). In practice, however, applicants in such cases sometimes choose to deposit in order to avoid all risk of their application being rejected on

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the grounds of insufficient disclosure. Some applicants, on the other hand, prefer to take this risk. In cases where the microorganism is already generally available from a culture collection, the situation is perhaps more straightforward. Some countries (e.g. Germany, USA, USSR) require the applicant to furnish a declaration signed by the culture collection and stating that the organism in question is in fact available and will remain available for the period dictated by the relevant national law. In this connection, it is worth noting that the USA in some cases presently requires such a declaration - at least for deposits made outside the USA - even where an organism has been deposited under the Budapest Treaty. As mentioned earlier, the Japanese patent office will recognise the availability of strains from culture collections outside Japan only if they have been deposited under the Budapest Treaty. 6.5

Practical considerations So far this chapter has been concerned with the principles of biotechnological patents and the requirements of various countries; now the essentially practical aspects of the patenting process must be considered. To use specific examples, either actual or hypothetical, for this purpose would give too narrow a picture. Therefore, the more general question of seeking patent protection for an unspecified invention involving the use of a new (i.e. not already generally available) microorganism will be considered. 6.5.1

The patent agent (patent attorney)

The job of the patent agent is, put simply, to obtain a patent on behalf of the applicant. The agent's knowledge and understanding of patent procedures world-wide are essential to guide the applicant through the complex business of seeking patent protection, helping to draft the technical description, formulating the claims, dealing with the patent authorities, ensuring deadlines are met and so on. However, his technical knowledge of the invention and its background cannot be expected to equal that of the inventor, who must therefore be prepared to spend time and effort in familiarising him with every aspect. In his turn, the agent can often offer valuable advice on areas where more experimental work might be done before filing in order to make the application as strong as possible. The importance of these considerations is shown by the fact that many large firms have full-time patent agents on their staff to ensure that their inventions are adequately

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protected. The small organisation and the academic inventor, therefore, are well advised to obtain the services of a professionally qualified patent agent if they are considering applying for patent protection. 6.5.2

Disclosing the invention

Premature disclosure. As mentioned earlier, making a full disclosure of an invention is the applicant's side of the bargain that will give him a legal monopoly, and is a fundamental prerequisite for obtaining a patent. However, the disclosure must be made in the proper way and at the right time. Above all the invention must not be disclosed prematurely, for its novelty (see Section 6.2.2 above) will be assessed by most patent offices in the light of what is already known (the state of the art) on the day the patent application is filed. The prevailing state of the art includes any contributions the applicant himself may have made to it, whether orally, by visual display, or by display or sale of a product. Thus to avoid premature disclosure, the academic inventor in particular must abjure the normal practices of discussing his findings with other workers or publishing them in scientific journals until he has filed his patent application. Information revealed by breach of the applicant's confidence does not jeopardise a patent application, but since breach of confidence is often difficult to prove, the wisest course is to make no disclosure until the application has been safely filed. These strictures do not wholly apply (the exact conditions vary) in relation to those few countries, notably the USA, that allow a 'grace period' (see Section 6.2.2 above). However, even here it must be remembered that although the relevant national law may allow disclosure during a grace period preceding the basic national filing, such a disclosure will be prejudicial to subsequent foreign filings. The patent specification - technical description. The patent specification

contains the written disclosure or technical description of the invention and the patent claims, which state the scope and kinds of monopoly being asked for. The precise wording of the patent specification is of great importance and it is here that the skill of the patent agent comes into play in ensuring that the description (supplemented by deposit see Section 6.5.5 below) fulfils the requirements of disclosure and that the claims are drafted to afford the best protection. The technical description is exactly that; the invention is described in detail in scientific and technical terms - it is not addressed to the

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layman - and put into the context of the field to which it applies, the problems it aims to solve and the way in which solutions are achieved. The preferred format of the description is well established and typically includes the following: field, background, object and summary of the invention, followed by the detailed description of the invention. Crespi (1982) has discussed the layout of the technical description more fully, with actual examples, and the reader is referred to his book for more information. As far as the present account is concerned, the first point is that the description must clearly convey the novelty, inventiveness and industrial applicability of the invention, and must describe the methodology in sufficient detail (worked examples being usual, but not mandatory) to enable a skilled person to reproduce the invention for himself and show that it works in accordance with the claims of the inventor. Second, the technical description must also describe any new microorganism involved in the invention. Clearly the accession number assigned to the organism by the culture collection in which it has been deposited must be quoted, but beyond that the extent of characterisation required varies between countries. The most extensive requirements are those of the Japanese patent office, which gives in its 'examination standard' a detailed list of the properties which should be recorded. The EPO has less stringent guidelines, and at the other extreme the Netherlands will accept deposit of the organism in lieu of any characterisation. Many countries expect the kind of taxonomic data that would be used in scientific publications, although most do not insist on it and accept deposit as a means of offsetting deficiencies in the written characterisation. In general, an applicant is well advised to provide characterisation data 'to the extent available to him' (Crespi, 1985). The patent specification - claims. The claims are perhaps the most important part of the patent specification as far as the applicant is concerned, because they set out precisely the extent of the protection being sought. This is particularly so in, for example, the UK and USA where great attention is paid to the exact wording of the claims. Any loophole left here can leave the inventor exposed to competition from which he might otherwise have been protected. In Germany, on the other hand, the claims are viewed less literally in that more attention is given to them as indicators of the basic inventive idea. The EPO adopts a middle course, trying to strike a balance between the rights of the inventor and those of third parties. Again, the reader is referred to Crespi (1982)

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and to Ruffles (1986) for more detailed discussion about patent claims; only the salient points will be given here. Normal practice is to make the scope of the initial claims as broad as possible, leaving it to the patent office to object if it believes that too much is being claimed for the invention. In the final event, the applicant may feel that the degree of protection he has been allowed is less than it ought to be, but this is better than finding that he is the author of his own misfortune by having claimed too little in the first place. Nevertheless, the claims must not be extravagant; they must be based on the description and be supported by it. Thus the greater the degree of novelty and ingenuity indicated by the description, and the wider the variety of worked examples given, then the broader the claims that are likely to be accepted. Claims are usually presented as a numbered set. The first is often the broadest and is the general claim; this is followed by subclaims, each defining by example particular aspects of the general claim, and each generally being narrower in scope than the one before it. The subclaims represent fall-back positions if the broader claims are held invalid. Claims of more than one kind should be included wherever possible, e.g. a new chemical compound, a microbiological process of producing it, the organism used in the process claimed per se, a method of diagnosis using the new compound, and a kit incorporating the new compound and conventional reagents for the diagnosis. Table 6.4 gives examples of sets of patent claims relating to different cell types. Table 6.4. Examples of sets of patent claims US Patent no. 4,567,146

We claim: 1. A recombinant plasmid characterized in that it contains DNA of (1) a first Rhizobium plasmid identifiable as being the same as the plasmid pVW5JI or pVW3JI of lower molecular weight present in the culture of the strain of Rhizobium leguminosarum NCIB 11685 or 11683 respectively and (2) a second Rhizobium plasmid found in bacteria of another strain of Rhizobium leguminosarum, said second plasmid having Rhizobium genes coding for nodulation, nitrogen fixation and hydrogen uptake ability but which is non-transmissible. 2. A method of preparing a culture of bacteria of the genus Rhizobium, which method is characterized in that (1) in a first cross, a donor strain of Rhizobium, containing (a) a Rhizobium plasmid lacking genes coding for nodulation but which is transmissible, is crossed with a recipient strain of Rhizobium, carrying (b) a Rhizobium plasmid having Rhizobium genes coding for nodulation, nitrogen fixation and hydrogen uptake ability but which is non-transmissible, whereby a

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Table 6.4 (cont.) transconjugant strain carrying a plasmid which is formed from said plasmids (a) and (b) and is a conjugal precursor of a recombinant plasmid (c) having genes coding for nodulation, nitrogen fixation and hydrogen uptake ability and being transmissible is obtained; (2) said transconjugant strain is separated from donor and recipient strains and cultured to produce a substantially pure culture thereof; (3) in a second cross, the transconjugant strain from the first cross is used as a donor strain and crossed with a plasmid-containing recipient strain whereby a transconjugant strain carrying a recombinant plasmid (c) is obtained; and (4) said transconjugant strain from the second cross is separated from donor and recipient strains and cultured to produce a substantially pure culture thereof. 3. A method according to claim 2 characterized in that the transmissible plasmid (a) carries at least one drug-resistance gene. 4. A method according to claim 3 characterized in that the transmissible plasmid is pVW5JI or pVW3JI, identifiable as being the same as the plasmid of lower molecular weight present in the culture of a strain of Rhizobium leguminosarum NCIB 11685 (pVW5JI) or NCIB 11683 (pVW3JI), and a kanamycin-resistant transconjugant strain is separated in each cross. 5. A method according to claim 2 characterized in that the transmissible plasmid (a) contains a selectable determinant. 6. A method according to claim 2, characterized in that the donor and recipient strain are of the species Rhizobium leguminosarum. 7. A method of impairing hydrogen uptake ability to bacteria of the genus Rhizobium, which method is characterized in that (1) a strain of Rhizobium leguminosarum NCIB 11684 or NCIB 11682, as a donor strain, is crossed with a recipient strain of Rhizobium leguminosarum to produce a kanamycin-resistant transconjugant strain, said recipient strain being one which permits selection of the transconjugant strain against the donor and recipient strains and which allows the transconjugant strain to be selected against when used as a donor in a subsequent cross with another strain of Rhizobium leguminosarum, (2) said transconjugant strain is separated from the donor and recipient strains and cultured to produce a substantially pure culture thereof; (3) in a second cross the transconjugant strain obtained from the first cross is used as a donor strain and crossed with a recipient strain of Rhizobium leguminosarum to produce a kanamycin-resistant transconjugant strain and (4) said transconjugant strain from the second cross is separated from the donor and recipient strains to produce a biologically pure culture thereof. 8. A method according to claim 7 characterized in that the recipient strain for the first cross is auxotrophic and has resistance to a drug other than kanamycin. 9. A method according to claim 7 or 8 wherein the recipient strain for the second cross is a naturally occurring strain. 10. A Rhizobium plasmid pIJ1008 having Rhizobium genes coding for streptomycin and kanamycin resistance, nodulation, nitrogen fixation and hydrogen uptake properties, which is transmissible and which is the plasmid of lowest molecular weight present in the culture of a strain of Rhizobium

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Table 6.4 (cont.) leguminosarum NCIB 11684 by virtue of the fact that it migrates the fastest on agarose gel in a gel electrophoresis determination in which a gel of 0.7% agarose in Tris-borate buffer of pH. 8.3 is subjected to electrophoresis at 25 mA and 100 volts at 4°C for 16 to 20 hours in the dark. 11. A Rhizobium plasmid pIJ1007 having Rhizobium genes coding for streptomycin and kanamycin resistance, nodulation, nitrogen fixation and hydrogen uptake properties, which is transmissible and which is the plasmid of lowest molecular weight present in the culture of a strain of Rhizobium leguminosarum NCIB 11682 by virtue of the fact that it migrates the fastest on agarose gel in a gel electrophoresis determination in which a gel of 0.7% agarose in Tri-borate buffer of pH 8.3 is subjected to electrophoresis at 25 mA and 100 volts at 4°C for 16 to 20 hours in the dark. 12. A biologically pure culture of bacteria of the genus Rhizobium characterized in that it contains a plasmid selected from the group consisting of pIJ1008 and pIJ1007. 13. A culture according to claim 12 of bacteria of the species Rhizobium leguminosarum. 14. A biologically pure culture of bacteria of the genus Rhizobium containing a recombinant plasmid characterized in that said plasmid contains DNA of (1) a first Rhizobium plasmid identifiable as being the same as the plasmid pVW5JI or lower molecular weight present in the culture of the strain Rhizobium leguminosarum NCIB 11685 or 11683 respectively and (2) a second Rhizobium plasmid found in bacteria of another strain of Rhizobium leguminosarum, said second plasmid having Rhizobium genes coding for nodulation, nitrogen fixation and hydrogen uptake ability but which is non-transmissible. US Patent no. 4,546,082

What is claimed is: 1. A DNA expression vector capable of expressing in yeast cells a product which is secreted from said yeast cells, said vector comprising at least a segment of alpha-factor precursor gene and at least one segment encoding a polypeptide. 2. A DNA expression vector according to claim 1 wherein said segment encoding a polypeptide is an insertion into said alpha-factor precursor gene. 3. A DNA expression vector according to claim 1 wherein said segment encoding a polypeptide is a fusion at a terminus of said alpha-factor precursor gene. 4. A DNA expression vector according to claims 2 or 3 wherein coding sequences for mature alpha-factor are absent from said segment of alpha-factor precursor. 5. A DNA expression vector according to claim 1 wherein said polypeptide is somatostatin. 6. A DNA expression vector according to claim 1 wherein said polypeptide is 7. A DNA expression vector according to claim 1 wherein said polypeptide is an enkephalin. 8. A yeast strain transformed with a DNA expression vector of claim 1.

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Table 6.4 (cont.) 9. A method for producing a DNA expression vector containing alpha-factor gene comprising the steps of (a) transforming a MAT alpha 2 leu 2 yeast strain with a gene bank constructed in plasmid YEpl3; (b) selecting for leu transformants from the population formed in step (a); (c) replacing the transformants from step (b) and (d) screening for alpha-factor producing colonies. 10. A DNA expression vector formed according to the method of claim 9. UK Patent no. 1,346,051

What we claim is: 1. Fusarium graminearum Schwabe deposited with the Commonwealth My cological Institute and assigned the number I.M.I. 145425 and variants and mutants thereof. 2. Fusarium graminearum Schwabe 1-7 deposited with the Commonwealth Mycological Institute and assigned the number I.M.I. 154209. 3. Fusarium graminearum Schwabe 1-8 deposited with the Commonwealth Mycological Institute and assigned the number I.M.I. 154211. 4. Fusarium graminearum Schwabe 1-9 deposited with the Commonwealth Mycological Institute and assigned the number I.M.I. 154212. 5. Fusarium graminearum Schwabe 1-15 deposited with the Commonwealth Mycological Institute and assigned the number I.M.I. 154213. 6. Fusarium graminearum Schwabe 1-16 deposited with the Commonwealth Mycological Institute and assigned the number I.M.I. 154210. 7. Fungal cultures containing a strain of Fusarium graminearum Schwabe I.M.I. 145425 or a mutant or variant thereof in a culture medium in which this strain is present in a culture medium containing or being supplied with nutrients or additives necessary for the sustenance and multiplication of the strain, the medium having a pH between 3.5 and 7 and the temperature of the medium being maintained at a precise value within the range of between 25 and 34°C. 8. A method of cultivating a strain of Fusarium graminearum Schwabe I.M.I. 145425 or a mutant or variant thereof wherein the strain is present in a culture medium containing or being supplied with nutrients or additives necessary for the sustenance and multiplication of the strain, the medium having a pH between 3.5 and 7 and the temperature of the medium being maintained at a precise value within the range of between 25 and 34°C, 9. A method for the preparation of variants of Fusarium graminearum Schwabe I.M.I. 145425 which comprises growing the parent strain I.M.I. 145425 under continuous culture conditions with carbon limitation in a fermentation. 10. A method for the preparation of variants of Fusarium graminearum Schwabe I.M.I. 145425 which comprises growing the parent strain I.M.I. 145425 on a glucose based medium at 25° to 30°C under continuous culture conditions at a dilution rate of 0.10 to 0.15 hrs" 1 with carbon limitation in a fermentation for 1100 hours. 11. A method as claimed in claim 10 wherein the resulting proliferated variants are isolated by dilution plating. 12. A method for the preparation of variants of Fusarium graminearum

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Table 6.4 (cont.) Schwabe I.M.I. 145425 substantially as described with reference to Examples 1 to 5 hereinbefore set forth. 13. Fungal cultures containing Fusarium graminearum Schwabe I.M.I. 145425 or mutants or variants thereof substantially as described with reference to any one of Examples 6 to 11 hereinbefore set forth. UK Patent no. 1,300,391

What we claim is: 1. A human embryo liver cell line having the characteristics of cells deposited with the American Type Culture Collection under number CL99. 2. A cell culture system comprising cells derived from the human embryo liver cell line designated by A.T.C.C. number CL99 in a nutrient culture medium therefore. 3. A virus culture system comprising cells derived from the human embryo liver cell line designated by A.T.C.C. number CL99 inoculated with a virus capable of replication in said cells, and a nutrient culture medium adapted to support growth of the virus-cell system. 4. A culture system according to claim 2 or 3, wherein the nutrient culture medium contains Eagle's minimum essential medium and heat-inactivated foetal calf serum. 5. A culture system according to claim 4, wherein the nutrient culture medium contains Eagle's minimum essential medium, heat-inactivated foetal calf serum, sodium bicarbonate and one or more antibiotics. 6. A virus cultivation process which comprises maintaining a viable culture of cells derived from the human embryo liver cell line designated by A.T.C.C. number CL99 in a nutrient culture medium, inoculating the culture with a virus to which the cells are susceptible and cultivating the virus in the culture. 7. A process according to claim 6, wherein the virus is of the group consisting of adenoviruses, San Carlos viruses, ECHO viruses, arthropodborne group A viruses and other arboviruses, pox viruses, myxoviruses, paramyxoviruses, piconaviruses, herpes viruses and the AR 17 haemovirus. 8. A process according to claim 6, wherein the virus is a hepatitis virus. 9. A process according to claim 7, wherein the virus is of the group consisting of adenovirus types 2, 3, 4, 5, 7 and 17, San Carlos virus types 3, 6, 8 and 49, ECHO virus type 11, Sindbis virus, vaccinia virus, influenza A2 virus and other influenza viruses, Sabin poliovirus type 1 and other poliomyelitis viruses, and the AR-17 haemovirus. 10. A virus whenever cultivated by the process of any of claims 6 to 9. 11. Antigenic material obtained from a virus according to claim 10. 12. A vaccine comprising a virus according to claim 10, in an administrable form and dosage. 13. A vaccine comprising antigenic material according to claim 11, in an administrable form and dosage. 14. A vaccine comprising antibodies produced by a virus according to claim 10 or antigenic material according to claim 11, in an administrable form and dosage. 15. A cell culture system substantially as described in Example 3. 16. A virus cultivation process substantially as described in Example 3.

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6.5.3

Filing the application

A single application filed in one country will result in patent protection only in that country. Therefore when the disclosure of an invention is likely to lead to serious foreign competition the normal course is to seek protection in several countries. Fortunately this does not have to be done all at once and the first application is usually filed in the applicant's own country. The patent office gives this application a number and, more importantly, a filing date. The significance of this first filing date ('priority date') is that it establishes the priority of the invention; in other words any later applications made in that country by other people for the same invention are pre-empted by it. Furthermore, provided that the applicant files any corresponding foreign applications within 12 months of his basic national filing, the original priority date is also recognised by nearly all overseas countries. However, the same priority date cannot be claimed for material not included in the basic national application (the 'priority document'). The EPO also recognises the original priority date for applications filed with it within 12 months of the basic national filing. The advantage of the European system is that an application filed with the EPO results in a clutch of 'national' patents, valid in those countries party to the EPC that the applicant has designated as being territories in which he wants patent protection. The applicant must, however, designate these countries at the outset; he can drop some of them later by not paying renewal fees, but he cannot add to them. Use of the EPC system is not mandatory in Europe however; if an inventor wishes, he can instead file separate national applications in individual countries. In fact, if protection is not required in more than two or three European countries, the national route may be cheaper. The first steps along the road to obtaining patent protection involve drafting the patent specification, filing the basic national application and, within 12 months, filing the appropriate foreign applications (redrafting and developing further the original specification if appropriate). As Crespi (1982) has pointed out, a year is not long when account is taken of the need to evaluate the importance of the invention, decide on the extent of foreign patenting desired and implement the decision. Implementation involves drafting the final specification, sending documents around the world and possibly having the specification translated into other languages. In this last respect it is all too easy, say, for the English-speaking applicant to forget that the German Patent Office will expect an application to be written in German.

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(When filing with the EPO, however, the application may be drafted in English, French or German.) Thus although the patent agent will take care of all the documentary procedures, consulting the applicant where necessary, the latter cannot afford to relax completely at this stage. Attention must also be paid, of course, to ensuring that by the filing date (or, where applicable, the priority date) the microorganism used in the invention has been deposited in a suitable culture collection. This will be discussed in considerable detail later (see Section 6.5.5 below); for the present it is more convenient to follow the progress of the patent application itself. 6.5.4

Patent office procedures

Once the final national and/or foreign applications have been filed and the microorganism deposited, the applicant must wait for his application to be processed by the patent office. The exact procedures and the time they take vary widely between countries. Therefore, only a broad outline, illustrated with a few examples, can be given here. All major patent offices carry out a novelty search, which is usually a patent and literature search, followed by a critical or substantive examination of the application. Under the EPC and many European national systems, the search and examination are treated separately. After the search, a report is sent to the patent agent, pointing out material (including earlier patents) considered relevant to the application. Under the EPC, this report and the patent application itself should be published 18 months after the priority (basic national filing) date, although in practice, delays in the issuance of the search report are common. At this point, the claims can be modified by the applicant if it seems unlikely that they will be accepted as they stand. Also with the publication of the application, the deposited microorganism becomes available (to varying extents) under most European systems (see Release of samples in Section 6.4.2 above). For the application to proceed further, the applicant must now ask for a substantive examination to be made of it. This request must be made within a specified period or the application will lapse. Under the Japanese system, patent applications are published after 18 months, but there is no search or examination unless and until the applicant requests it, which he must do within seven years. In Japan and the USA, both the search and substantive examination are carried out before a report is issued to the applicant. In all systems, a written response to the patent examiner's report

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must be made within a certain period or the application may fail by default. There usually then follows a variable period of negotiation with the examiner ('prosecution of the application') as to how broad the final claims should be in view of the prior art. If agreement is reached, then the application is accepted. If not, it is refused and the applicant and his agent must then consider whether to pursue an appeal to a higher tribunal. Negotiations with the patent authorities and the meeting of deadlines are usually taken care of by the patent agent. Unless questions of a highly technical nature are raised, the involvement of the inventor himself in these proceedings is generally minimal. After an application has been accepted, in most countries the patent is then granted and published (for the first time in the USA and Canada). At this point the deposited microorganism becomes available for the first time in the USA, Canada, Japan and (unless the EPC route has been followed) the Netherlands. Under the EPC system the microorganism, available since the first publication only to an independent expert if the applicant has so opted, becomes generally available. Major patent offices permit a period immediately after the patent application has been allowed or the patent has been granted for it to be challenged by third parties. The extent of this 'opposition' period varies considerably between countries. Thus, for instance, the EPC allows a nine month period for after-grant revocation of a European patent (before it becomes a collection of national patents). Japan allows an opposition period of three months before grant. In the USA there is no opposition procedure as such. Instead, anyone can ask for a reexamination of any patent, regardless of when it was granted, provided that he can cite pertinent prior art previously unconsidered by the Patent Office and which is sufficient to convince the Office that the issue should be re-opened. Exceptionally, a UK patent can be revoked by application to the Patent Office at any time after grant. The length of time for which a patent lasts once it has been granted also varies between different countries. In the USA and Canada, for instance, the period is 17 years from the date of grant, regardless of how long the application has been pending the outcome of negotiations between the applicant and the patent office. In Europe, on the other hand, the term is 20 years from the application date. In Japan, it is 15 years from publication for opposition purposes, or 20 years from the application date, whichever is the shorter. Maintenance of the

Patent protection for biotechnological inventions

121

patent for its full term is subject to periodic renewal fees, non-payment of which will result in the patent lapsing. 6.5.5

Depositing the microorganism

With an invention involving the use of a new microorganism, that is, one not already available to the public, there is one other vital act to be performed at an early stage of the patenting procedure. The microorganism must be deposited in a suitable culture collection in order to complete the disclosure of the invention. Since in almost all cases the deposit must have been effected at the latest by the filing date (or, where applicable, by the priority date), it might fairly be said that, apart from drafting the specification, this is often the first practical step to be taken towards obtaining the patent. Moreover, it is a step that relies for its effective accomplishment much more on the inventor than on his agent. The latter can do little more than advise about the documentary formalities and deadlines and perhaps suggest appropriate collections. It is the inventor who knows his organism, the technical difficulties in handling it, how long is needed to grow it, and any legal constraints in respect of its pathogenicity, which might delay matters. Thus it is up to the inventor to brief his agent so that between them they can ensure that the culture collection receives the organism in good time to allow for any possible delays or mishaps. As mentioned above, the mechanism of deposit is now regulated internationally by the Budapest Treaty, and even in countries not yet party to the Treaty, its procedures tend to be viewed as a model system. Nevertheless, for purely national purposes, deposit under the Treaty is often not necessary (see National deposit requirements in Section 6.4.2 above). However, for the international recognition of a single deposit, using the Budapest Treaty is by far the safest course of action and the following account will be concerned mainly with the Budapest Treaty system. Although the following discussion goes into some detail, much more comprehensive information is contained in the Guide to the Deposit of Microorganisms for the Purposes of Patent Pro-

cedure issued by the World Intellectual Property Organization (WIPO), Geneva. For convenience, the term 'depositor' will be used in this connection in preference to 'applicant' or 'inventor'. Lastly, it should be borne in mind throughout that the date of deposit is the date on which the culture collection physically receives the culture, rather than the date when the culture is formally accepted.

122

I. /. Bousfield

Requirements of the Budapest Treaty. Under the Budapest Treaty a deposit

must be made with an International Depositary Authority (IDA) according to the provisions of Rule 6 of the Treaty. The requirements for making such a deposit are laid down in Rule 6.1(a), which requires that the culture sent to an IDA must be accompanied by a written statement, signed by the depositor and containing the following information: (i) an indication that the deposit is made under the Treaty and an undertaking not to withdraw it for the period specified in Rule 9.1; The period specified in Rule 9.1 is five years after the latest request for a sample, and in any case at least 30 years. The important thing to note here is that a deposit made under the Budapest Treaty is permanent and having made it, the depositor cannot later ask for it to be cancelled, regardless of whether a patent is eventually granted. This applies even if he abandons his patent application. (ii) the name and address of the depositor; (iii) details of the conditions necessary for the cultivation of the microorganism, for its storage and for testing its viability and also, where a mixture of microorganisms is deposited, descriptions of the components of the mixture and at least one of the methods permitting the checking of their presence; This requirement simply ensures that the culture collection is given enough information to enable it to handle the organism correctly. The instructions about (intentionally) mixed cultures are included so that a positive viability statement (see below) is not issued when all the components of the co-culture are not viable. (iv) an identification reference (number, symbols etc.) given by the depositor to the microorganism; The term 'identification reference' is sometimes wrongly taken to refer to a taxonomic identification, whereas it simply means 'strain designation'. (v) an indication of the properties of the microorganism which are or may be dangerous to health or the environment, or an indication that the depositor is not aware of such properties. The requirements of Rule 6.1(a) are mandatory and cannot be varied either by the depositor or by the IDA. Indeed if the depositor does not comply with them all, the IDA is obliged to ask him to do so before it can accept the deposit. The same does not apply to Rule 6.1(b), which

Patent protection for biotechnological inventions is not really a rule at all but simply an exhortation. According to Rule 6.1(b) 'it is strongly recommended that the written statement . . . should contain the scientific description and/or proposed taxonomic designation of the deposited microorganism'. As well as the above requirements, the Treaty permits the IDA to set certain conditions of its own (Rule 6.3(a)). These are: (i) that the microorganism be deposited in the form and quantity necessary for the purpose of the Treaty and these Regulations; Thus an IDA may require that cultures are submitted to it in a particular state, e.g. freeze-dried, in agar stabs, etc., and that a specified number of replicates is provided. (ii) that a form established by such authority and duly completed by the depositor for the purposes of the administrative procedures of such authority be furnished; This refers to the accession form (and any other form) routinely used by the culture collection. (iii) that the written statement . . . be drafted in the language, or in any of the languages, specified by such authority . . . This is an obvious requirement, permitting a Japanese depository, for example, to ask for information to be supplied to it in Japanese, (iv) that the fee for storage . . . be paid; (v) that, to the extent permitted by the applicable law, the depositor enter into a contract with such authority defining the liabilities of the depositor and the said authority. This provides for the IDA to make the kind of contractual arrangements with the depositor that would be usual under the laws of contract of the IDA's own country. Without this provision, some culture collections would have been unwilling to become IDAs. It is entirely up to the IDA whether it requires any or all of the above from the depositor, but if it does, then the depositor has no option but to comply. Some of the requirements of existing IDAs are summarised in Table 6.5. These, then, are the official requirements which the depositor must meet. For its part, the IDA also must fulfil certain obligations under the Treaty. In particular it must issue to the depositor an official receipt (the contents of which are laid down by the Treaty) stating that it has received and accepted the deposit and it must, as soon as possible, test the viability of the culture deposited and issue an official statement to the depositor informing him of the result. If the culture proves not to

123

Table 6.5. International Depositary Authorities at 30 November 1989

International Depositary Authority

Microorganisms accepted

SUMMARY

Agricultural Research Service Culture Collection (NRRL) Peoria USA American Type Culture Collection (ATCC) Rockville USA Australian Government Analytical Laboratory (AGAL) Pymble NSW Australia Centraalbureau voor Schimmelcultures (CBS) Baarn Netherlands Collection Nationale de Cultures de Microorganismes (CNCM) Paris France

Non-pathogenic bacteria, actinomycetes, yeasts, moulds

Most kinds

Non-pathogenic bacteria, actinomycetes, yeasts and fungi; non-hazardous nucleic acid preparations and phages

Fungi, yeasts, actinomycetes, bacteria

Bacteria, actinomycetes, fungi, yeasts, viruses, animal and plant cells

Minimum no. of replicates to be provided by the depositor

Culture Collection of Algae and Protozoa (CCAP) Ambleside and Oban UK Culture Collection of the CAB International Mycological Institute (CMI CC) Kew UK Deutsche Sammlung von Mikroorganismen (DSM)*7 Braunschweig Federal Republic of Germany European Collection of Animal Cell Cultures (ECACC) Fermentation Research Institute (FRI) Ibaraki-ken, Japan Institute of Microorganism Biochemistry and Physiology of the USSR Academy of Science (IBFM) Moscow Region, USSR In Vitro International Inc. (IVI) Linthicum, USA National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) Sofia, Bulgaria

Algae, non-pathogenic protozoa

Non-pathogenic fungi

Non-pathogenic bacteria, actinomycetes, fungi, yeasts, phages, plasmids in a host or as a DNA preparation Cell lines, animal viruses Non-pathogenic fungi, yeasts, bacteria, actinomycetes, animal and plant cell cultures Non-pathogenic bacteria including actinomycetes, fungi including yeasts

Most kinds Bacteria including actinomycetes, microscopic fungi and yeasts, algae, animal cells, animal viruses, plasmids

Table 6.5. (cont.) International Depositary Authority

Microorganisms accepted

National Collection of Agricultural & Industrial Microorganisms (NCAIM) Budapest, Hungary National Collections of Industrial and Marine Bacteria Ltd. (NCIMB) Aberdeen, UK National Collection of Type Cultures (NCTC) London, UK National Collection of Yeast Cultures (NCYC) Norwich, UK Nationale Sammlung von Mikroorganismen (IMET) Jena, German Democratic Republic USSR Research Institute for Antibiotics of the USSR Ministry of the Medical and Microbiological Industry (VNIIA) Moscow, USSR

Non-pathogenic bacteria, fungi, yeasts

Non-pathogenic bacteria, actinomycetes, yeasts, phages, plasmids in host cells or as DNA preparations, seeds Pathogenic bacteria Non-pathogenic yeasts Non-pathogenic bacteria including actinomycetes and cyanobacteria, fungi including yeasts, algae, bacterial viruses, plasmids in hosts or as DNA preparations Non-pathogenic bacteria including actinomycetes, fungi including yeasts

Minimum no. of replicates to be provided by the depositor

USSR Research Institute for Genetics and Industrial Microorganism Breeding of the USSR Ministry of the Medical and Microbiological Industry (VNII Genetika) Moscow, USSR

Non-pathogenic bacteria including actinomycetes, fungi including yeasts

DETAILED INFORMATION Australia

Australian Government Analytical Laboratories (AGAL) The New South Wales Regional Laboratory 1 Suakin Street Pymble, NSW 2073

Bacteria (including actinomycetes), yeasts and fungi with a hazard categorisation no greater than WHO Classification Risk Group 2, that can be preserved without significant change to their properties by the methods of preservation in use (liquid nitrogen storage and lyophilisation). Nucleic acid preparations and phages may be accepted if the depositor certifies that they pose no hazard when handled by normal laboratory procedures and the depositor supplies suitable material for preservation. At present, AGAL does not accept for deposit animal, plant, algal and protozoal cultures, cultures of viral, rickettsial and chlamydial agents, microorganisms prohibited by Australian law, or fastidious microorganisms which may require in the view of the curator special attention to handling and preparation for storage.

Bacteria, fungi, yeasts: 6 Phages, plasmids: sufficient quantity and titre for preservation

Table 6.5. (cont.)

International Depositary Authority

Microorganisms accepted

Bulgaria National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) 125 Lenin Blvd. Block 2 Sofia

Bacteria, actinomycetes, microscopic fungi, yeasts, microscopic algae, animal cell lines, animal viruses and microorganisms containing plasmids.

France Collection Nationale de Cultures de Micro-organismes (CNCM) Institut Pasteur 28 rue du Dr Roux F-75724 Paris Cedex 15

Minimum no. of replicates to be provided by the depositor Bacteria, fungi, yeasts: 3 Viruses, cell lines: 10

Bacteria (including actinomycetes), bacteria Cell lines: 12 containing plasmids; filamentous fungi and Other organisms: 8 yeasts, and viruses, EXCEPT: - cellular cultures (animal cells, including hybridomas and plant cells): - microorganisms whose manipulation calls for physical insulation standards of P3 or P4 level, according to the information provided by the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules and Laboratory Safety Monograph; - microorganisms liable to require viability testing that the CNCM is technically not able to carry out; - mixtures of undefined and/or unidentifiable microorganisms. The CNCM reserves the possibility of refusing any microorganism for security reasons: specific risks to human beings, animals, plants and the environment.

In the eventuality of the deposit of cultures that are not or cannot be lyophilised, the CNCM must be consulted, prior to the transmittal of the microorganism, regarding the possibilities and conditions for acceptance of the samples; however, it is advisable to make this prior consultation in all cases. Federal Republic of Germany DSM - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg lb D-3300 Braunschweig

Bacteria, including actinomycetes, fungi, including yeasts, bacteriophages, plasmids (a) in a host (b) as an isolated DNA preparation. The following phytopathogenic microorganisms are not accepted for deposit: Coniothyrium fagacearum; Endothia parasitica; Gloeosporium ampelophagum; Septoria musiva; Synchytrium endobioticum.

German Democratic Republic IMET - Nationale Sammlung von Mikroorganismen IMET-Hinterlegungsstelle Beutenbergstrasse 11 6900 Jena

Strains of bacteria, including actinomycetes and cyanobacteria, fungi, including yeasts, unicellular and filamentous algae, bacterial viruses, plasmids per se or included in strains. Strains and materials constituting a danger for man's health or a hazard for the environment, or for the storage or maintenance of which the depository authority is technically not in a position, may be excluded from deposit.

Plasmids: 5 Other organisms: 2

Table 6.5. (cont.) International Depositary Authority

Minimum no. of replicates to be provided by the depositor

Microorganisms accepted

Hungary

National Collection of Agricultural and Industrial Microorganisms (NCAIM) Department of Microbiology and Biotechnology University of Horticulture and the Food Industry Somloi ut 14-16 H-1118 Budapest

3 or 25fl

Bacteria (including Streptomyces) except obligate human pathogenic species (e.g., Corynebacterium diphtheriae, Yersinia pestis, etc.).

Mycobacterium

leprae,

Fungi, including yeasts and moulds, except some

pathogens

(Blastomyces,

Coccidioides,

Histoplasma, etc.), as well as certain basidiomycetous and plant pathogenic fungi which cannot be preserved reliably. Apart from the above-mentioned, the following may not, at present, be accepted for deposit: - viruses, phages, rickettsiae; - algae, protozoa; - cell lines, hybridomas.

Japan

Fermentation Research Institute (FRI) Agency of Industrial Science and Technology Ministry of International Trade and Industry 1-3, Higashi 1-chome Tsukuba-shi Ibaraki-ken 305

Fungi, yeasts, bacteria, actinomycetes, animal cell cultures and plant cell cultures, EXCEPT: - microorganisms having properties which are or may be dangerous to human health or the environment; - microorganisms which require the physical containment level P3 or P4 For experiments, as described in the Prime Minister's Guidelines for Recombinant DNA Experiments of 1986.

5

Netherlands Centraalbureau voor Schimmelcultures (CBS) Oosterstraat 1 Postbus 273 NL-3740 AG Baarn United Kingdom Commonwealth Agricultural Bureau (CAB), International Mycological Institute (CAB IMI) Ferry Lane Kew, Surrey TW9 3AF Culture Collection of Algae and Protozoa (CCAP) FRESHWATER BIOLOGICAL ASSOCIATION

Windermere Laboratory The Ferry House Far Sawrey Ambleside, Cumbria LA22 OLP and

Fungi, including yeasts; actinomycetes, bacteria other than actinomycetes.

Fungal isolates, other than known human and animal pathogens and yeasts, that can be preserved without significant change to their properties by the methods of preservation in use. (i) Freshwater and terrestrial algae and freeliving protozoa (Freshwater Biological Association); and (ii) marine algae, other than large seaweeds (Scottish Marine Biological Association).

SCOTTISH MARINE BIOLOGICAL ASSOCIATION

Dunstaffnage Marine Research Laboratory PO Box 3 Oban, Argyll PA34 4AD European Collection of Animal Cell Cultures (ECACC) Vaccine Research and Production Laboratory Public Health Laboratory Service Centre for Applied Microbiology and Research Porton Down Salisbury, Wiltshire SP4 OJG

Cell lines that can be preserved without significant change to or loss of their properties by freezing and long-term storage; viruses capable of assay in tissue culture. A statement on their possible pathogenicity to man and/or animals is required at the time of deposit. Up to and including ACDP Category 3* can be accepted for deposit. * Advisory Committee on Dangerous Pathogens: Categorisation of Pathogens according to Hazard and Categories of Containment, ISBN 0/11/883761/3, HMSO, London.

12

Table 6.5. (cont.) International Depositary Authority

Microorganisms accepted

National Collections of Industrial and Marine Bacteria Ltd (NCIMB) 23 St Machar Drive Aberdeen AB2 1RY

(a) Bacteria including actinomycetes, that can be preserved without significant change to their properties by liquid nitrogen freezing or by freeze-drying (lyophilisation), and which are allocated to a hazard group no higher than Group 2 as defined by the UK Advisory Committee on Dangerous Pathogens (ACDP). (b) Plasmids, including recombinants, either (i) cloned into a bacterial or actinomycete host, or (ii) as naked DNA preparations. As regards (i), above, the hazard category of the host with or without its plasmid must be no higher than ACDP Group 2. As regards (ii), above, the phenotypic markers of the plasmid must be capable of expression in a bacterial or actinomycete host and must be readily detectable. In all cases, the physical containment requirements must not be higher than level II as defined by the UK Advisory Committee on Genetic Manipulation (ACGM) and the properties of the deposited material must not be changed significantly by liquid nitrogen freezing or freeze-drying. (c) Bacteriophages that have a hazard rating and containment requirements no greater than

Minimum no. of replicates to be provided by the depositor Bacteria, yeasts, phages: 2 Plasmids (as DNA): 10 ml at 20 mcg/ml Seeds: 2500

those cited in (a) or (b), above, and which can be preserved without significant change to their properties by liquid nitrogen freezing or by lyophilisation. (d) Yeasts (including those containing plasmids) that can be preserved without significant change to their properties by liquid nitrogen freezing or by freeze-drying, that are allocated to a hazard group no higher than ACDP Group 2, and which require physical containment no higher than level IIACGM. (c) Seeds that can be dried to a low moisture content and/or stored at low temperatures without excessive impairment of germination potential. The right is reserved to refuse the deposit of seeds where dormancy is exceptionally difficult to break. The acceptance of seeds by NCIMB and the furnishing of samples thereof are subject at all times to the provisions of the Plant Health (Great Britain) Order 1987, including any future amendments or revisions of that Order. NCIMB must be notified in advance of all intended deposits of seeds so that it may ensure that all relevant regulations are complied with. Any seeds received without prior notification may be destroyed immediately. Notwithstanding the foregoing, NCIMB reserves the right to refuse to accept any material for deposit which in the opinion of the Curator presents an unacceptable hazard or is technically too difficult to handle.

Table 6.5. (cont.) International Depositary Authority

National Collection of Type Cultures (NCTC) Central Public Health Laboratory 61 Colindale Avenue London NW9 5HT National Collection of Yeast Cultures (NCYC) AFRC Institute of Food Research Norwich Laboratory Colney Lane Norwich NR4 7UA USA Agricultural Research Service Culture Collection (NRRL) 1815 North University Street Peoria, Illinois 61604

Minimum no. of replicates to be provided by the depositor

Microorganisms accepted In exceptional circumstances, NCIMB may accept deposits which can only be maintained in active culture, but acceptance of such deposits, and relevant fees, must be decided on an individual basis by prior negotiation with the prospective depositor. Bacteria that can be preserved without significant change to their properties by freezedrying and which are pathogenic to man and/or animals.

1

Yeasts other than known pathogens that can be preserved without significant change to their properties by freeze-drying or, exceptionally, in active culture.

1

1. All strains of agriculturally and industrially important bacteria, yeasts, molds and

Ito30*

Actinomycetales, EXCEPT: (a) Actinobacillus (all species);

(anaerobic/microaerophilic,

Actinomyces

all

species);

Arizona (all species); Bacillus anthracis; Bartonella (all species); Bordetella (all spe-

ties); Borrelia (all species); Brucella (all species); Clostridium botulinum; Clostridium chauvoei; Clostridium haemolyticum} Clostridium histolyticum; Clostridium novyi; Clostridium septicum; Clostridium tetani; Corynebacterium diphtheriae; Corynebacterium equi; Corynebacterium haemolyticum; Corynebacterium pseudotuberculosis; Corynebacterium pyogenes; Corynebacterium renale; Diplococcus (all species); Erysipelothrix (all species); Escherichia coli (all enteropathogenic types); Frandsella (all species); Haemophilus (all species); Herellea (all species); Klebsiella (all species); Leptospira (all species); Listeria (all species); Mima (all species); Moraxella (all species); Mycobacterium avium; Mycobacterium bovis) Mycobacterium tuberculosis) Mycoplasma (all species); Neisseria (all species); Pasteurella (all species); Pseudomonas pseudomallei) Salmonella (all species); Shigella (all species); Sphaerophorus (all species); Streptobacillus (all species); Streptococcus (all pathogenic species); Treponema (all species); Vibrio (all species); Yersinia (all species); (b) Blastomyces (all species); Coccidioides (all species); Cryptococcus neoformans; Cryptococcus uniguttulatus; Histoplasma (all species); Paracoccidioides (all species). (c) All viral, Rickettsial, and Chiamydial agents.

Table 6.5. (cont.) International Depositary Authority

Microorganisms accepted (d) Agents which may introduce or disseminate any contagious or infectious disease of animals, humans or poultry and which require a permit for entry and/or distribution within the United States of America. (e) Agents which are classified as plant pests and which require a permit for entry and/or distribution within the United States of America. (f) Mixtures of microorganisms. (g) Fastitious microorganisms which require (in the view of the Curator) more than reasonable attention in handling and preparation of lyophilised material. (h) Phages not inserted in microorganisms, (i) Monoclonal antibodies, (j) All cell lines. (k) Plasmids not inserted in microorganisms. 2. Recombinant strains of microorganisms, strains containing recombinant DNA molecules, strains containing their own naturally occurring plasmid(s), strains containing inserted naturally occurring plasmid(s) from another host, strains containing inserted constructed plasmid(s), and strains containing viruses of any kind, excluding those already

Minimum no. of replicates to be provided by the depositor

American Type Culture Collection (ATCC) 12301 Parklawn Drive Rockville, Maryland 20852

listed as non-acceptable, only if the deposit document accompanying the microbial preparation(s) includes a clear statement that progeny of the strain(s) can be processed at a Physical Containment Level of PI or less and Biological Containment requirements meet all other criteria specified by the US Department of Health and Human Services, National Institutes of Health; Guidelines for Research Involving Recombinant DNA Molecules, December 1978 (Federal Register, Vol. 43, No. 247 - Friday, December 22, 1978) and any subsequent revisions. Algae, animal embryos, animal viruses, bac- Animal viruses, cell lines, teria, cell lines, fungi, hybridomas, oncogenes, naked plasmids: 25 plant viruses, plasmids, plant tissue cultures, Other organisms: 6 phages, protozoa, seeds, yeasts. Seeds: 2500 The ATCC must be informed of the physical containment level required for experiments using the host vector system, as described in the 1980 National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules

(i.e. PI, P2, P3 or P4 facility). The ATCC, for the time being, will accept only those hosts containing plasmids which can be worked in a PI or P2 facility. Certain animal viruses may require viability testing in an animal host, which the ATCC may be unable to provide. In such case, the deposit cannot be accepted. Plant viruses which cannot be mechanically inoculated also cannot be accepted.

Table 6.5. (cont.) Minimum no. of replicates to be provided by the depositor

International Depositary Authority

Microorganisms accepted

In Vitro International, Inc. (IVI) 611(P) Hammonds Ferry Road Linthicum, Maryland 21090

Algae, bacteria with plasmids, bacteriopha- Bacteria, fungi, yeasts: 3 ges, cell cultures, fungi, protozoa, animal and Other organisms: 6 plant viruses and seeds. Recombinant strains of Seeds: 400 microorganisms will also be accepted, but IVI must be notified in advance of accepting the deposit of the physical containment level required for the host vector system, as prescribed by the National Institutes of Health Guidelines. At present, IVI will accept only hosts containing recombinant plasmids that can be worked in a PI or P2 facility.

USSR Institute of Microorganism Biochemistry and Physiology of the USSR Academy of Science (IBFM) Pushchino-na-Oke 142292 Moscow Region

USSR Research Institute for Antibiotics of the USSR Ministry of the Medical and Microbiological Industry (VNIAA) Nagatinskaya Street 3-a 113105 Moscow

USSR Research Institute for Genetics and Industrial Microorganism Breeding of the USSR Ministry of the Medical and Microbiological Industry (VNII Genetika) Dorozhnaya Street No. 8 113545 Moscow

Bacteria (including actinomycetes) and microscopic fungi (including yeasts), also if they are carriers of recombinant DNA, are accepted for deposit, to the exclusion of microorganisms that cause disease in man and animals and microorganisms that have a toxicogenic effect on plants or require them to be quarantined. Bacteria (including actinomycetes) and microscopic fungi (including yeasts) for essentially medical purposes are accepted for deposit, to the exclusion of microorganisms that cause disease in man and animals and microorganisms that are toxicogenic for plants or require them to be quarantined. Bacteria (including actinomycetes) and microscopic fungi (including yeasts) for essentially industrial and non-medical purposes are accepted for deposit, to the exclusion of microorganisms that cause disease in man and animals and microorganisms that have a toxicogenic effect on plants or require them to be quarantined.

If depositor's own lyophilised cultures are to be stored and distributed.

5 or 50"

5 or 50"

140

I. /. Bousfield

be viable, the deposit is worthless, which can lead to major problems (see below). The IDA must also keep the deposit secret from all except those entitled to receive samples; it must maintain the deposit for the 30 or more years required by the Treaty, checking the viability 'at reasonable intervals' or at any time on the demand of the depositor; it must supply cultures to anyone entitled under the relevant patent law to receive them (provided that the IDA has been given proof of entitlement - see below); it must inform the depositor when and to whom it has released samples; it must be impartial and available to any depositor under the same conditions. New deposits. If by some mischance a microorganism which was viable when deposited dies during storage, or if indeed for any reason the IDA can no longer supply cultures of it, then the IDA must notify the depositor immediately. The latter then has the option of replacing it (Article 4), and provided he does so within three months, the date on which the original deposit was made still stands. When making a new deposit, the depositor must (under Rule 6.2) provide the IDA with: (1) a signed statement that he is submitting a culture of the same microorganism as deposited previously; (2) an indication of the date on which he received notification from the IDA of its inability to supply cultures of the previous deposit; (3) the reason he is making the new deposit; (4) a copy of the receipt and the last positive viability statement in respect of the previous deposit; (5) a copy of the most recent scientific description and/or taxonomic designation submitted to the IDA in respect of the previous deposit; (6) if the new deposit is being made with a different IDA, all the indications required under Rule 6.1 (a) (see above). With regard to item (6) above, the new deposit can be made with a different IDA if the original IDA is no longer operating as such (either entirely or just in respect of that particular kind of microorganism) or if import/export regulations render the original IDA inappropriate for that particular deposit. It must be remembered that the provisions for making a new deposit cannot be applied to a microorganism which was shown by the IDA to be non-viable when it was originally deposited. There must have been at least one positive viability statement.

Patent protection for biotechnological inventions

141

'Converted' deposits. The Budapest Treaty allows (Rule 6.4(d)) for a deposit made outside its provisions to be 'converted' to a Treaty deposit (provided, of course, that the culture collection holding the deposit is an IDA). Where the microorganism was deposited before the culture collection became an IDA the date of deposit of the 'conversion' is held to be the date on which the collection acquired IDA status. Otherwise the date of deposit is the date on which the collection physically received the culture. The procedure for converting a deposit usually involves completing the same forms as are used for making a deposit de novo. However, only the original depositor (or his successor) can convert a deposit. In all other cases, a separate deposit of the same organism must be made under the Treaty. Conversion is a useful facility because it means that an earlier nonBudapest Treaty deposit can be accorded the international recognition which it might not otherwise command. Conversion is essential for the recognition by the Japanese patent office of any non-Budapest Treaty deposit made outside Japan. Guidelines for deposit. It is generally recognised that many depositors (and sometimes their patent agents) are unlikely to be familiar with the minute details of the Budapest Treaty and may not be aware of their obligations in respect of it. Therefore, the forms which ID As ask prospective depositors to fill in are generally so designed that by completing them correctly the depositor automatically provides all the information required of him by the Treaty. These forms vary to some extent between ID As, but they all follow a similar general pattern. Any IDA will supply specimens of its forms on request. Making a deposit under the Budapest Treaty should be quite straightforward, but problems can and do arise. It has to be said that many of these are of the depositor's (or his agent's) own making and that they can be avoided by adhering to a few simple guidelines. Perhaps the most important thing to be remembered is that the Budapest Treaty procedures take a certain amount of time to complete, even when they are operating ideally. Thus although in principle a deposit does not have to reach the IDA until the filing (or priority) date of the relevant patent application, in practice the wise depositor will start the depositing procedure in good time to allow for any possible delays. Thus if he is intending to deposit in a foreign IDA, say, he should bear in mind any import or quarantine regulations. For instance, it can take several weeks, or even months, to obtain a permit

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to import cell lines and viruses into the USA. Last-minute deposits are unwise for several reasons, some of the most common being: (1) postal delays: the culture fails to arrive in time; (2) customs delays: with deposits from overseas, depositors have not provided adequate shipping information; (3) the deposit is not the kind of microorganism accepted by the IDA (see Table 6.5); (4) the microorganism cannot be recovered from the package, e.g. because the culture tube is broken; (5) the deposit proves to be non-viable; if a microorganism is found by the IDA from the outset to be non-viable, the original date of deposit cannot be applied to any replacement (see above). For most bacteria, fungi, yeasts, algae and protozoa, viability testing usually takes 3-5 days; for animal cell lines a week or slightly longer is normal; and for animal viruses and plant tissue cells, up to a month is not unusual. It cannot be emphasised too strongly that however good the depositor's intentions may be, patent offices recognise only the actuality of the deposit. With all this in mind the prudent depositor will also pay attention to a few more elementary points to ensure a timely and trouble free deposit. He will ensure that the microorganism he wishes to deposit is one of the kinds that the IDA he had chosen can officially accept under the Budapest Treaty (see Table 6.5). If there are likely to be technical problems with the organism he will advise the IDA in advance. He will check the administrative and technical requirements of the chosen IDA and ask for the appropriate patent deposit forms, which he will then fill in completely and correctly, for by doing so he should automatically comply with the requirements of Rule 6.1(a) (see above). Although Rule 6.1(a) states that the microorganism should be accompanied by a written statement (the completed deposit form), in practice it is often helpful to an IDA to receive the written information in advance of the microorganism itself, so that arrangements can be made to deal with the deposit promptly. This is particularly helpful if, say, a special growth medium has to be prepared by the IDA. Lastly, if the depositor's patent agent is likely to be communicating with the IDA, the depositor should let the IDA know, otherwise it may withhold information until it has ascertained the agent's right to receive it. Depending on its policy and on the kind of material being deposited,

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an IDA may or may not prepare subcultures for eventual distribution. Thus in the case of cell lines and naked plasmids (not cloned into a host), for instance, the depositor is usually required to supply sufficient material for the IDA to distribute direct. On the other hand, for bacteria, yeasts, moulds, etc. (with or without plasmids) it is more usual for the IDA to distribute its own preparations. In this case, many IDAs will ask the depositor to check the authenticity of their preparations - a fairly normal culture collection practice. The depositor is not obliged by the Budapest Treaty to check these preparations, but he is well advised to do so to ensure that the cultures to be sent out by the IDA will in fact do what is claimed for them in the patent application. The official aspects of the depositing procedure end with the issuing of the receipt and viability statement by the IDA. These are important for they are the documentary proof that on a particular date a viable deposit has been made according to the terms of the Budapest Treaty. Technically the receipt should be issued first, but in practice many IDAs find it more convenient to await the results of the viability test and then send out the receipt and viability statement together. In general, for deposits of most bacteria, fungi, yeasts, algae and protozoa, the depositor could expect an IDA to send him both documents within a few days of it having received the deposit. For animal cell lines a week or slightly longer would be normal, and for animal viruses and plant tissue cells four or five weeks would be more usual. 6.5.6

Obtaining a sample of a patent deposit

So far in this account the deposit procedure has been considered primarily from the viewpoint of the depositor. It would be useful now to look briefly at the procedures which a third party must follow in order to obtain a culture, since the whole point of deposit is to make the microorganism available. It is generally admitted that culture collections can neither be expected to be familiar with the patent laws of countries throughout the world nor to know what stage patent applications relating to the deposits they hold have reached. Thus to require a collection to judge for itself whether a particular person is legally entitled to a culture of a particular deposit is considered by many to impose an unfair burden on the collection. Therefore the Budapest Treaty attempts to place the onus on patent offices to ensure that ID As are not put in this position (Rule 11.3). Patent offices in countries whose laws require that deposited microorganisms must be available without restriction to

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anyone once the relevant patents have been granted and published can notify the IDAs from time to time of the accession numbers of the strains cited in these patents. However, this provision is not usually adopted. The US Patent Office, for instance, directs that a microorganism must be available from the date of issuance of the relevant US patent, but it does not advise the IDA of this date. Since the issuance or not of a US patent is a simple matter of fact, in the event of any request an IDA merely has to ascertain this fact, either from the requesting party or the depositor. In the author's experience this does not cause any major problems, although the actual furnishing of the sample may be delayed slightly while evidence of publication is obtained. The IDA can then meet any requests for the strains in question without the need for evidence of entitlement. In cases where the availability of the microorganism is restricted and/or where evidence of entitlement to receive a sample is required, anyone requiring a culture must either obtain the written authorisation of the depositor or he must obtain from a relevant patent office a certificate stating: (1) that a patent application in respect of the strain in question has actually been filed with that office; (2) whether the application has been published; (3) that the person requesting the culture is legally entitled to receive it and has met any conditions that the law requires. On receipt of a request accompanied by such a certificate, or by the written authorisation of the depositor, the IDA will supply the culture (subject to its normal fee for such cultures being paid). At the same time, the IDA will inform the depositor when and to whom it has supplied the culture, as it is obliged to do by Rule 11.4(g) of the Treaty, unless the depositor has specifically waived this right to be informed. Except where the direct authorisation of the depositor has been sought, the request for a culture must be made on an official form which can be had from the patent office(s) with which the relevant application has been filed. Most ID As also have copies of these forms. Thus, the procedure for obtaining a culture of a microorganism deposited under the Budapest Treaty is: (1) ask the appropriate patent office, or the IDA, for a copy of the form to be used for requesting samples of microorganisms deposited under the Budapest Treaty; (2) complete the part of the form to be filled in by 'the requesting party';

Patent protection for biotechnological inventions (3) send the entire form to the patent office, not to the IDA; (4) when the form bearing the appropriate stamp of authorisation is received back from the patent office, send it to the IDA along with a normal purchase order. Procedures for obtaining organisms deposited for patent purposes outside the Budapest Treaty vary according to the national law. In such cases, the culture collection will have been informed by the depositor or the patent office of the appropriate requirements and should be able to advise accordingly. It must be remembered that the procedures outlined above relate only to the right to receive cultures according to patent law. They do not override any requirements to be met in respect of import and quarantine regulations, health and safety procedures, plant disease regulations, etc. Thus as well as obtaining patent office authorisation, a person requesting a culture must also ensure that he has obtained any permit or licence necessary for handling the organism in question. 6.6

Further reading Adler, R. G. (1984). Biotechnology as an intellectual property. Science 224, 357-63. Anonymous (1982). Japanese Patent Office guidelines for examination of inventions of microorganisms. Yuasa and Hara Journal 9 (3). Beier, F. K., Crespi, R. S. & Straus, J. (1985). Biotechnology and Patent Protection: An International Review. Paris: Organization for Economic

Co-operation and Development. Biggart, W. A. (1981). Patentability in the United States of microorganisms, processes utilizing microorganisms, products produced by microorganisms and microorganism mutational and genetic information techniques. IDEA, Journal of Law and Technology 22, 113-36. Budapest Treaty (1981). Budapest Treaty on the International recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure 1977 and

Regulations 1981. Geneva: World Intellectual Property Organization. Byrne, N. J. (1979). Patents on life. European Intellectual Property Review 1,

279-300. Byrne, N. J. (1983). The agritechnical criteria in plant breeders' rights law. Industrial Property (1983), 294-303. Convention (1980). Convention of the Grant of European Patents. (European

Patent Convention) 1973 with 1980 amendments. Munich: European Patent Organization. Cooper, I. P. (1982). Biotechnology and the Law. New York: Clark Boarman Co. Crespi, R. S. (1981). Biotechnology and patents - past and future. European Intellectual Property Review 3, 134-40.

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I. J. Bousfield Crespi, R. S. (1982). Patenting in the Biological Sciences. Chichester: John Wiley & Sons. Crespi, R. S. (1985a) Biotechnology patents - a case of special pleading? European Intellectual Property Review 7, 190-3. Crespi, R.S. (1985b) Microbiological inventions and the patent law - the international dimension. Biotechnology and Genetic Engineering Reviews 3, 1-37. Crespi, R. S. (1985c) Patent protection in biotechnology: questions, answers and observations. In Biotechnology and Patent Protection, ed. F. K. Beier, R. S. Crespi & J. Straus, pp. 36-^85. Paris: Organization for Economic Co-operation and Development. Crespi, R. S. (1986). Patent issues in biotechnology. In Biotechnology and Crop Improvement and Protection, British Crop Protection Council Monograph No. 34, ed. Peter R. Day, pp. 209-17. In re Diamond & Chakrabarty (1980). US Patents Quarterly 206, 193. Duffy, J. I. (1980). Chemicals by Enzymatic and Microbial Processes. Recent Advances. Park Ridge, New Jersey: Noyes Data Corp. Halluin, A. P. (1982). Patenting the results of genetic engineering research: an overview. In Patenting of Life Forms, Banbury Report No. 10, pp. 67126. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Hiini, A. (1977). The disclosure in patent applications for microbiological inventions. International Review of Industrial Property and Copyright Law 8, 500-21. Hiini, A. & Buss, V. (1982). Patent protection in the field of genetic engineering. Industrial Property (1982), 356-68. International Convention (1978). International Convention for the Protection of New Varieties of Plants 1961, revised 1972. Geneva: World Intellectual Property Organization. Irons, E.S. & Sears, M. H. (1975). Patents in relation to microbiology. Annual Review of Microbiology 29, 319-32. In re Lundak (1985). US Patents Quarterly 227, 90. Plant, D. W., Reimers, N. J. & Zinder, N. D. (eds.) (1982). Patenting of Life Forms. Banbury Report No. 10. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Pridham, T. G. & Hesseltine, C. W. (1975). Culture collections and patent depositions. Advances in Applied Microbiology 19, 1-23. Straus, J. (1985). Industrial Property Protection of Biotechnological Inventions: Analysis of Certain Basic Issues. Document BIG/281. Geneva: World Intellectual Property Organization. Teschemacher, R. (1982). Patentability of microorganisms per se. International Review of Industrial Property and Copyright Law 13, 27-41. Wegner, H. C. (1979). Patenting the products of genetic engineering. Biotechnology Letters 1, 145-40 and 193. Wegner, H. C. (1980). The Chakrabarty decision patenting products of genetic engineering. European Intellectual Property Review 2, 304-7. WIPO (1980) Records of the Budapest Diplomatic Conference for the Conclusion of a Treaty on the International Recognition of the Deposit of Microorganisms for

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the Purposes of Patent Procedure. WIPO Publication no. 332 (E), p. 119. Geneva: World Intellectual Property Organization. WIPO (1986). Report adopted by the 2nd session of the Paris Union Committee of Experts on Biotechnological Inventions and Industrial Property. WIPO

Document BioT/CE/II/3. Geneva: World Intellectual Property Organization. WIPO (1988). Guide to the Deposit of Microorganisms for the Purposes of Patent Procedure.

The author gratefully acknowledges the helpful comments and criticisms made by many colleagues during the writing of this chapter. Particular thanks go to Mrs B. A. Brandon of the American Type Culture Collection and Mr R. S. Crespi of the British Technology Group. A singular debt of gratitude is owed to Mr R. K. Percy of the British Technology Group, for his extensive advice and painstaking correction of the original draft.

Culture collection services D. ALLSOPP and F. P. SIMIONE

7.1

Introduction In response to the needs of users, many culture collections provide a range of services to the scientific, technological and commercial world. This chapter provides an introduction to the types of services available from culture collections, but it is beyond its scope to give a comprehensive list of such services. As the range of work that can be undertaken is increasing at many of the collections, the reader should contact individual collections to find out whether they can offer particular services. 7.2

Types of services

7.2.1

Directly associated and customer services

The two major services which are intrinsically part of culture collection work are those concerning the identification and preservation of organisms. Collections of necessity need expertise in these fields to be able to function, and many provide comprehensive services in these areas. Aspects of culture identification methods (Chapter 5), sales of cultures (Chapter 3), preservation techniques (Chapter 4) and patent deposits (Chapter 6) are covered elsewhere in this volume. Safe-Deposits. Many collections hold organisms which are not listed in their catalogues. These cultures are held for a variety of reasons: the organisms may not be fully identified, their taxonomic status may be unclear, their stability in preservation may be suspect or they may be held at the request of the depositor who wishes to have back-up material and yet retain ownership and confidentiality, not releasing the strain to other parties. Many collections have introduced safe-deposit 148

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services as a back-up to the depositor's working collection, providing a service intermediate between an open collection deposit and a deposit for patent purposes. Such services enable the depositor to have important organisms professionally preserved and maintained even if the collection would not normally be interested in accessioning them. There is obvious merit in the safekeeping of cultures while they are the subject of research, especially as many laboratories do not have optimum preservation facilities. Most collections make a charge for safe-deposits to cover the long-term storage costs and quality control procedures that are required. Advice on strain selection. Collections are able to give advice on the selection of strains for special purposes. Such services may involve collection staff in a considerable amount of work and are limited by the sources of information available. In the past such services have relied upon the individual expertise of collection staff and their personal knowledge of the scientific literature. With the development of computer databases and strain data networks (Chapter 2), such services are becoming more frequent and are increasing in efficiency. However, as databases are searched electronically and appropriate microorganisms selected, the expertise of the collection staff is still needed to draw attention to closely allied genera and species that might merit study or to point out the idiosyncracies of individual strains. These advisory services are being placed on a more formal basis and charges may be made. Advice on maintenance of organisms. Most collections are able to provide information on preservation systems, either on a formal or informal basis. Some of the major collections have produced substantial publications covering the preservation of their own groups of organisms and these may be consulted. However, if any doubt exists or if different media are being tried, the collections can always be consulted for advice. Advisory sheets on particular preservation techniques, or related topics such as the handling of pathogens, elimination of contamination or mite infestation, are often available on request. Biochemical services. Some groups of organisms, such as bacteria and yeasts, are identified using biochemical tests, and such methods are increasingly used for other organisms, particularly the filamentous fungi. The need for taxonomic clarification using biochemical criteria

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goes hand in hand with an increasing requirement to provide metabolic and other physiological data to users of the collections. Applied biological industries, and in particular biotechnology, increasingly seek organisms on the basis of activity rather than name, and collections are responding to this requirement. Collections now enhance their catalogues with strain data, and supply information to strain databases and computer networks as a routine procedure. Some collections also carry out custom-designed screening programmes to select strains with specified attributes for individual clients, particularly in the fields of enzyme and secondary metabolite (including toxin) production or specific growth requirements. These biochemical services serve both as directly associated and contract services of culture collections (see also Research and development work in section below). 7.2.2

Contract services

The services outlined in Section 7.2.1 above usually exist as a consequence of the collection's normal activities; however, in recent years there has been an expansion in other services offered by collections for a variety of reasons. For example, many collections are associated with other institutions, such as research organisations, taxonomic institutes, educational institutions, or commercial firms, any of which may make specific demands on the services of the collection. Again, the collection may be supported by funds provided from external sources which may require particular expertise or services to be developed. Many collections are currently under financial pressure to increase their earning potential, and this has stimulated the development of income-generating activities. Biological testing. Many modern standards and specifications require the use of microorganisms and cell lines in the testing of products. These include the mould growth resistance of materials (Fig. 7.1), the testing of disinfectants against a range of bacteria, the assessment of mutagenicity of materials against a range of organisms, and toxicity testing. For economic and other reasons there is strong pressure to move away from the use of live animals in the testing of products and towards the use of microorganisms or cell lines instead. These tests can be carried out in any suitably equipped laboratory, but culture collections are in a very favourable position to carry out

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such testing themselves, using the organisms or cell lines which they would normally supply to outsiders for such work; indeed, collections may be identified in the standards themselves as an approved source Fig. 7.1. Chamber used for the testing of industrial products for resistance to mould growth at the CAB International Mycological Institute (IMI).

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of such material. Unless a company is equipped for routine testing as part of its quality control procedures, it is often more cost effective for such work to be carried out in a specialised laboratory. To establish a laboratory and train staff to carry out extensive biological testing infrequently would be very expensive, and not necessarily satisfactory from a technical point of view. However, an economic service can be offered by culture collections equipped to carry out such work on a regular basis. Examples of testing standards using microorganisms and cell lines include British (BS), European (ISO) and USA (ASTM, USP) standards for fungal resistance testing, sterility testing, preservative effectiveness, toxicity and biocompatibility testing (Table 7.1). Culture collections provide reference cultures used in clinical laboratory standards procedures (e.g. NCCLS, ECCLS), and can provide uniform, quality assured sets of reference cultures for use in on-site biological testing for both clinical and industrial applications.

Table 7.1. Examples of testing standards involving microorganisms, cell cultures and related materials British Standards BS 1982 Methods of testing for fungal resistance of manufactured building materials made of, or containing, materials of organic origin BS 2011 The environmental testing of electronic components and electronic equipment. Test J. Mould growth BS 28458 Flexible insulating sleeving for electrical purposes. Section 12. Mould growth BS 3046 Specification for adhesives for hanging flexible wall coverings. Appendix G. Test for susceptibility to mould growth BS 4249 Specification for paper jointing. Section 5.7. Resistance to mould growth BS 5980 Specification for adhesives for use with ceramic tiles and mosaics. Section 7. Resistance to mould growth BS 6009 Wood preservatives. Determination of toxic values against wood destroying Basidiomycetes on an agar medium BS 6085 Methods of testing for the determination of the resistance of textiles to microbiological deterioration European Standards

ISO 846

Plastics - determination of behaviour under the action of fungi and bacteria - evaluation or measurement of change in mass or physical properties See also NFX41-514 and DIN 53739 for similar methods of plastics testing in France and Germany, respectively.

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Table 7.1. contd. US Standards ASTM D2574

Standard test method for resistance of emulsion paints in the container to attack by microorganisms ASTM D3273 Resistance to growth of mould on the surface of interior coatings in an environmental chamber. Standard test method for evaluating the degree of surface disfigurement of paint films by fungal growth or soil and dirt contamination ASTM G21-70 Standard recommended practice for determining resistance of synthetic polymeric materials to fungi ASTM G22-76 Standard recommended practice for determining resistance of plastics to bacteria FDA 21CFR610.12 Sterility testing of biological products FDA 21CFR610.30 Detection of mycoplasma contamination MIL STD 810D Environmental test methods. Method 508.2 Fungus NCCLS M2-A3 Performance standards for antimicrobial disk susceptibility tests NCCLS M7A Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically NCCLS Mil A Reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria USD A 9CFR113.26 Detection of viable bacteria and fungi in biological products USD A 9CFR113.28 Detection of Mycoplasma contamination USD A 9CFR113.29 Determination of moisture content in desiccated biological products USP Antimicrobial preservatives - effectiveness assay USP Microbial limits tests USP Sterility tests Consultancy. Many collections have staff with expertise in specialised areas commensurate with their collection responsibilities, who can be made available for consultancy work. Such work is often a forerunner of detailed investigations, or a research programme, and can be time consuming. Since collection staff time for this work is limited, consultancy work is usually offered on a fully charged basis. Research and development work. Culture collections equipped for testing work and consultancy work may also be involved in research and development work and in industrial investigations which require laboratory facilities. It is often difficult for collections to specify exactly what kind of work they would be prepared to accept, as many problems are unique and may be carried out on a one-off basis. It is usual, therefore, for collections to consider any type of work which falls in

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their general area of competence. By their very nature, culture collections usually have a wide range of scientific and industrial contacts, and even if they are not able to carry out particular investigations themselves, they may be able to advise on places where such work could be carried out. Culture collections can therefore act as referral centres and this function should not be overlooked. In addition to research topics on taxonomy and the preservation of organisms, major culture collections are often well placed to undertake other research, often allied to biotechnology, which may be initiated by short-term contracts and testing work. Potential topics for such longerterm research and development work might include: (1) screening large numbers of isolates for particular biochemical properties and end uses; (2) comparative studies in regard to enzyme or metabolite production of different strains of the same or closely related species; (3) development and evaluation of rapid detection procedures for compounds produced by organisms, including the development of commercial kits; (4) studies on the use of microorganisms as bio-control agents against insect pests, weeds and deteriogenic microorganisms; (5) selection of test organisms used in the evaluation of materials; (6) studies on growth requirements and the testing of bioreactors; (7) evaluation of media, culture vessels, diagnostic reagents and procedures. Custom preparations. Culture collections may be requested to provide bulk amounts of their organisms on an ad hoc basis to commercial organisations with limited facilities. Often this is an efficient and costeffective method of obtaining bulk inocula, since the collection has the expertise in growing the organisms and ready access to the media. Government agencies may request multiple units of mixtures of cultures for laboratory certification and proficiency testing in clinical laboratories. Manufacturers of diagnostic instruments may specify cultures with known properties for calibration of their instruments. Resource development. In the United States the Federal Government has used culture collections extensively to develop research reagents/

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cultures. These have been developed through joint efforts with the research scientists defining the needs and quality controls required and the banking and distribution aspects handled by the collections. 7.2.3

Confidentiality Work performed by culture collections can be carried out on a confidential basis if required. Formal mechanisms for ensuring confidentiality are in fact required by some laboratory accreditation schemes (see below) and are already in place in a number of collections. In some areas, such as identification services, users are sometimes content for material sent in to become part of the open collection, should the collection wish to retain it. However, in collections which deal with organisms of industrial importance, it may be more normal to carry out the work in confidence, with all material being destroyed after examination. Enquiries can be treated as confidential, and collections should be questioned about their policy and procedures regarding confidentiality. Submissions are a source of new organisms for building up the resources available from the collections, and some collections will identify cultures for a lower fee if the culture can be retained in the collection. 7.2.4

Laboratory accreditation In many countries national schemes exist for the accreditation of testing laboratories. Such schemes aim to establish standards for the accuracy and efficiency of measuring instruments, quality control procedures, record-keeping, and administration of the laboratory. Major companies may have their own accreditation schemes for laboratories that they use, but where national schemes exist, companies often accept the accreditation of the scheme and do not carry out individual laboratory accreditation on their own behalf. National schemes may also be recognised on an international basis and thus ease the way for laboratories to be accepted on a much wider geographical basis. There are now a sufficient number of national and international testing standards involving microorganisms to make such accreditation schemes worthwhile for culture collection laboratories to adopt. 7.3

Workshops and training Many culture collection staff are involved in educational activities, either because of their general scientific background or their detailed knowledge of culture collection practices. Instruction may be

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external, where staff are involved in training courses at local colleges, polytechnics or research institutes in their own country or overseas. Increasingly, however, collections are devising formal programmes of in-house courses. These may be arranged in direct response to a need identified either by the collection itself or by an outside body. In some parts of the world the educational system is such that credits towards a qualification can be gained by attendance at approved outside courses, and where such a system exists, it can ease the task of the culture collection in operating such courses. Courses vary in length from several weeks' or even months' duration (if dealing with topics such as identification), to one-day lecture courses or seminars on specific industrial or commercial topics. Outside speakers and instructors may be used to enable topics to be covered which extend beyond the expertise available in the collection itself. In addition to formal programmed and advertised courses, it is often possible to obtain individual training within culture collections to suit a particular need. Some collections have special facilities and staff devoted to training programmes, while others may use external facilities at educational institutions nearby. Charges are usually made for training, but in some cases the costs may be subsidised. Advice may be available from collections on suitable sources of funding for prospective students and trainees, especially from developing countries. Some culture collections are either owned by academic institutions such as universities, or are officially associated with them, enabling them to offer training towards MSc and PhD degrees by research. Some of the training offered is directly related to the normal activities of the collection, and instruction in preservation and maintenance techniques is often available; indeed, such training is often difficult to obtain from other sources. The training facilities offered by culture collections are often much greater than is generally known and the World Federation for Culture Collections' Education Committee is developing a list of teachers, their special expertise and courses available throughout the world (see Chapter 8). Individual collections may also provide information on training facilities in their scientific speciality or geographical area. 7.4

Publications, catalogues and publicity material The essential publication of any culture collection is its catalogue. Traditionally, these have been produced as hard-copy items but

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are now becoming available in a computerised on-line form (see Chapter 2). It is always worthwhile contacting a collection if an organism does not appear in its most recent hard-copy catalogue, as additional material may well be available, or information can be given on suitable organisms in reserve collections, which could possibly be released. In addition to catalogues, a few of the major collections produce scientific publications of their own (guides on preservation and maintenance, safety and handling, industrial uses and teaching) as well as articles in the scientific press and monographic studies. Details of such publications may be found in the collections' brochures or newsletters, or in bibliographic databases. Collection brochures are generally available free of charge, and it is worthwhile asking to be included on the collection's mailing list to ensure receipt of up-to-date information. Despite the fact that collections grow and change with time, enquirers or customers often rely on data from back issues of catalogues which may be many years old. This can lead to considerable confusion and users should ensure that they have the current catalogues before ordering cultures. These problems are minimised as catalogue and strain data become widely available through computer networks. For many people, however, printed information remains the most important reference material. 7.5

Fees and charges The ways in which culture collections are funded are extremely diverse. Very few culture collections exist as straightforward commercial entities; they are almost all subsidised in some fashion, either directly or indirectly, and the charges made for cultures do not reflect the true cost of production. Nevertheless, many collections offer discounts for bulk orders, special sets, or regularly ordered organisms such as those used for testing and teaching. The charges made for services, however, more accurately reflect the true cost, though they usually represent good value in comparison with totally commercial services, particularly in the areas of training. Charges for consultancy work, for testing and for laboratory services are normally at competitive commercial rates. Over the last few years, several important stimuli have been applied to culture collections, including the advent of biotechnology, the development of computerised databases and a harsher economic climate. These and other factors have led culture collections to examine

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the services they provide and to develop them to cater to the changing demand of their users. Expansion and diversification of the range of services has resulted. 7.6

Suggested reading Alexander, M., Daggett, P.-M., Gherna, R., Jong, S., Simione, F. & Hatt, H. (1980). American Type Culture Collection Methods. Laboratory Manual on Preservation: Freezing and Freeze Drying. Rockville, Maryland: American

Type Culture Collection. Allsopp, D. (1985). Fungal culture collections for the biotechnology industry. Industrial Biotechnology 5, 2. Allsopp, D. & Seal, J. J. (1986). Introduction to Biodeterioration, 136 pp. London: Edward Arnold. Batra, L. R. & Iijima, T. (eds) (1984). Critical Problems for Culture Collections,

71 pp. Osaka, Japan: Institute for Fermentation. Cour, I. G., Maxwell, G. & Hay, R. (1979). Tests for bacterial and fungal contaminants in cell cultures as applied at the ATCC. TCA Manual 5, 1157-60. Dilworth, S., Hay, R. & Daggett, P.-M. (1979). Procedures in use at the ATCC for detection of protozoan contaminants in culture cells. TCA Manual 5, 1107-10. Hawksworth, D. L. (1985). Fungus culture collections as a biotechnological resource. Biotechnology and Genetic Engineering Reviews 3, 417-53.

Hay, R. J. (1983). Availability and standardization of cell lines at the American Type Culture Collection: Current status and prospects for the future. In Cell Culture Test Methods, STP 810, ed. S. A. Brown, pp. 11426. Philadelphia: American Society for Testing and Materials. Jewell, J. E., Workman, R. & Zelenick, L. D. (1976). Moisture analysis of lyophilized allergenic extracts. In International Symposium on FreezeDrying of Biological Products. Developments in Biological Standardization 36,

181-9. Kelley, J. (1985). The testing of plastics for resistance to microorganisms. In Biodeterioration and Biodegradation of Plastics and Polymers ed. K. J. Seal,

pp. 111-24. Cranfield, UK: Cranfield Press. Kelley, J. & Allsopp, D. (1987). Mould growth testing of materials, components and equipment to national and international standards. Society of Applied Bacteriology, Technical Series 23. Oxford, UK: Blackwell

Scientific Publications. Lavappa, K. D. (1978). Trypsin-Giemsa banding procedure for chromosome preparations from cultured mammalian cells. TCA Manual 4, 761-1. Macy, J. (1978). Identification of cell line species by isoenzyme analysis. TCA Manual 4, 833-6. Macy, J. (1979). Tests for mycoplasmal contamination of cultured cells as applied at the ATCC. TCA Manual 5, 1151-5.

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May, M. C , Grim, E., Wheller, R. M. & West, J. (1982). Determination of residual moisture in freeze-dried viral vaccines: Karl Fischer, gravimetric thermogravimetric methodologies. /. Biological Standardization 10, 249-59.

8 Organisation of resource centres B. E. KIRSOP and E. J. DASILVA

8.1

Introduction Individual resource and information centres provide valuable services to biotechnology, but their role can be substantially enhanced if their activities are effectively co-ordinated. This has been recognised in the past, and a number of committees, federations and networks have been set up for this purpose at the national, regional and international levels. Although the origins and composition of existing organisations differ and their geographical locations are widespread, their common purpose is to support and develop the activities of resource and information centres for the benefit of microbiology. 8.2

International organisation

8.2.1

World Federation for Culture Collections

There are fewer difficulties in setting up national and regional co-ordinating mechanisms than international systems, and yet one of the first developments in this area was the formation of the World Federation for Culture Collections (WFCC). In 1962 at a Conference on Culture Collections held in Canada it was recommended that the International Association of Microbiological Societies (IAMS) set up a Section on Culture Collections. The Section was established in 1963. Five years later, at an International Conference on Culture Collections in Tokyo, the formation of the WFCC was proposed and an ad hoc committee, together with the Section on Culture Collections, drew up statutes which were agreed at a congress in 1970. Following the conversion of the IAMS to Union status, the WFCC is now a federation of the International Union of Microbiological Societies (IUMS) and an 160

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interdisciplinary Commission of the International Union of Biological Sciences (IUBS). The principal objective of the WFCC is to establish effective liaison between persons and organisations concerned with culture collections and the users of the collections both in the developed and developing regions of the world. To achieve this objective a structure of committees has been set up covering patents, postal and quarantine regulations, education, endangered collections, publicity and standards. Committee on Patent Procedures. The activities of the Committee on Patent Procedures have important implications for biotechnology. The procedures for patenting processes involving the use of microorganisms, animal or plant cells or of genetically manipulated organisms are described in Chapter 6. The various patent regulations existing in different parts of the world present a confusing picture to those wishing to take out patents, and professional guidance is essential. A number of organisations such as the World Intellectual Property Organisation (WIPO) are concerned with the rationalisation of the different systems, and the WFCC's patents committee has acted in an advisory capacity to them, providing microbiological input. Members of the Committee have attended WIPO meetings to advise on the implementation of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures. Additionally, they have monitored the functioning of the Treaty and provided evidence of difficulties that have arisen in its implementation. Recently a Guide to the Budapest Treaty has been published. Committee for Quarantine and Postal Regulations. The Committee for

Quarantine and Postal Regulations is similarly in close communication with the relevant postal regulatory bodies, such as the International Postal Union, National Postal Departments and the International Air Transport Association (IATA), and has put forward recommendations for the safe transport of infectious and non-infectious biological material (see Chapter 3). Members of the committee have been able to encourage international collaboration in this area by attending appropriate meetings and providing specialist advice in order to establish mechanisms for the safe transport of biological material throughout the world.

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Education Committee. The WFCC is aware of the lack of guidance given to students before finishing university training on the support and services available from the microbial resource centres of the world. A similar lack of general awareness exists among many working microbiologists in industry, research and education. Accordingly, the Education Committee of the WFCC has an on-going programme of activities to increase the amount of information on back-up available from culture collections. Projects include the publication of books, preparation of training videos, advisory leaflets, and the organisation of training courses, scientific symposia and international conferences. This present series of source books is part of the programme of the Education Committee, designed to increase the usefulness of culture collections to those working in biotechnology. A recent development has been the establishment of training schemes for individual scientists about to undertake extra responsibility in culture collections. Funds have been obtained from UNESCO and the International Union of Microbiological Societies to establish the first such scheme. Selected individuals have been given the opportunity to visit established collections to study administration and policy, 'shadowing' senior staff members and comparing different procedures and systems. It is expected that the scheme will lead to a greater awareness in young curators of the possibilities that exist in the establishment of new services and extended research initiatives. Committee for Endangered Collections. The Committee for Endangered

Collections is concerned to protect the microbial and cellular genetic resources of the world. Many of the major culture collections suffer from time to time from financial restrictions or from a change of direction in the interests of the host institute. Smaller collections are often transitory in nature and face difficulties on the retirement or relocation of the curator whose special interest the collection represents. The WFCC believes the conservation of these collections is of prime importance if the cultures and the substantial investment in terms of effort and expertise are not to be irretrievably lost. To enable emergency measures to be taken when difficulties arise, the Committee for Endangered Collections has obtained financial backing to assist in the provision of specialist, short-term support to allow the relocation of such collections to alternative laboratories willing and competent to take them over. The services of this committee may be used to provide advice to microbiologists who have developed collections of unique

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microorganisms during the course of their work, but who may not have the wish or expertise to maintain them in the long term or the resources to supply cultures to others. Publicity Committee. The WFCC's Publicity Committee plays a major role in the dissemination of information about the activities of the Federation to the microbiological community. It produces a newsletter at regular intervals and is closely involved with all administrative developments. In particular, it plays an important part in the fouryearly WFCC International Conference and in the preparation of posters for scientific conferences. The editor of the newsletter will consider the publication of appropriate material and welcomes information about meetings, publications and topics of general interest to members. Biotechnologists may use the newsletter as a forum for the discussion of issues - possibly controversial - that are of interest to fellow scientists. Typical of subjects that can usefully be discussed in the columns of the newsletter are questions relating to stable nomenclature of microorganisms, the retention of published strain designations, security measures for the release of potentially dangerous cultures to those unqualified to handle them or the rescue of important genetic resources. Committee for Standards. The WFCC is conscious of the fact that no authoritative standards exist for culture collections. Accordingly, a committee has been set up to prepare standards which can serve as guidelines. It is recognised that it is not possible for collections from different parts of the world to reach the same standards, and there is no intention to impose standards or categorise collections according to their facilities. It is nevertheless felt that the existence of WFCC Guidelines on Standards for Culture Collections will serve as a valuable reference index and a stimulus to attain the highest standards possible within the economic limits of any collection. Very high professional standards may be reached with modest resources. Data centres. In addition to the functions of these Committees, and others set up from time to time as the need arises, the WFCC has sponsored and is responsible for the World Data Center on Collections of Cultures of Microorganisms. The Center has pioneered the collection of data of this kind and has been responsible for the publication of

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three Directories listing the collections and the species they hold. The Center was originally housed in the University of Queensland's Department of Microbiology in Australia, but in 1986, on the retirement of its founder Director, was transferred to the Life Sciences Division at RIKEN, Tokyo, Japan. The WFCC is also co-sponsor with CODATA and IUMS of the international Microbial Strain Data Network (MSDN) set up to provide a referral system to the numerous data centres developing throughout the world listing microbial strain data. These two important activities of the WFCC, set up with international funding, are further discussed in Chapter 2. The WFCC plays a prime role in the organisation of culture collection activities internationally and has among its membership experts in many areas of microbiology. It exists to serve both the culture collections and their users and may be used as a powerful interdisciplinary organ of communication between biotechnologists and specialists in other areas of microbiology. 8.2.2

The MIRCEN Network

The UNESCO Courier of July 1975 carried a feature 'On the road to development - a UNESCO network for applied microbiology'. Therein several mechanisms - conferences, training courses and fellowships - were identified. Since then, as a means towards strengthening the world network, several regional and international initiatives have been built in through the establishment of microbiological resources centres (MIRCENs) (see Table 8.1). These are designed: (1) to provide the infrastructure for the building of a world network which would incorporate regional and interregional functional units geared to the management, distribution and utilisation of the microbial gene pool; (2) to strengthen efforts relating to the conservation of microorganisms with emphasis on Rhizobium gene pools in developing countries with an agrarian base; (3) to foster the development of new inexpensive technologies that are native to the region; (4) to promote the applications of microbiology in the strengthening of rural economies; (5) to serve as focal centres for the training of manpower and the imparting of microbiological knowledge. The first development in the UNESCO global network of Microbiological Resource Centres, consisting of centres in the developed world

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Table 8.1. Microbial resource centres Biotechnology MIRCENs

Ain Shams University, Faculty of Agriculture, Shobra-Khaima, Cairo, Arab Republic of Egypt Applied Research Division, Central American Research Institute for Industry (ICAITI), Ave La Reforma 4rA7, Zone 10, Apdo Postal 1552, Guatemala City, Guatemala. Bioconversion Technology MIRCEN, Caribbean Industrial Research Institute, Tunapuna, Trinidad and Tobago Bioengineering MIRCEN, Centre de Transfer de Microbiologie Biotechnologie, UPS-INSA, Avenue de Rangueil, F-31077 Toulouse Cedex, France Biotechnology MIRCEN, Department of Microbiology, University of Queensland, St Lucia, Queensland 4067, Australia CAB International Mycological Institute, Mycology MIRCEN, Ferry Lane, Kew, Surrey TW9 3AF, UK Department of Bacteriology, Karolinska Institutet, Fack, S-10401 Stockholm, Sweden Fermentation, Food and Waste Recycling MIRCEN, Thailand Institute of Scientific and Technoloigcal Research, 196 Phahonyothin Road, Bangken, Bangkok 9, Thailand Fermentation, Food and Waste Recycling MIRCEN, ICME, University of Osaka, Suita-shi 656, Osaka, Japan Institute for Biotechnological Studies, Research and Development Centre, University of Kent, Canterbury CT2 7TD, UK Marine Biotechnology MIRCEN, Department of Microbiology, University of Maryland, College Park Campus, Maryland 207742, USA Microbial Technology MIRCEN, Institute of Microbiology, Academia Sinica, Zhongguanoun, Beijing 100080, China Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), Avenida Belgrano y Pasaje Caseros, 4000 S.M. de Tucuman, Argentina University of Waterloo, Ontario, N2LK 3GI, Canada and University of Guelph, Guelph, Ontario NIG 2WI, Canada Rhizobium MIRCENs

Cell Culture and Nitrogen-Fixation Laboratory, Room 116, Building 011-A, Bare-West, Beltsville, Maryland 20705, USA Centre National de Recherches Agronomiques, d'Institut Senegalais de Recherches Agricoles, BP 51, Bambey, Senegal Departments of Soil Sciences and Botany, University of Nairobi, PO Box 30197, Nairobi, Kenya IPAGRO, Postal 776, 90000 Porto Alegre, Rio Grande do Sul, Brazil NIFTAL Project, College of Tropical Agriculture and Human Resources, University of Hawaii, PO Box 'O', Paia, Hawaii 96779, USA World Data Center MIRCEN

World Data Center on Collections and Microorganisms, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-01, Japan

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and regional networks in the developing countries, was the establishment of the World Data Center (WDC) on Microorganisms (see above and Chapter 2) in Queensland, Australia. The MIRCEN at the Karolinska Institute, Sweden, in addition to developing microbiological techniques for the identification of microorganisms at the WDC, has pioneered the organisation of a series of MIRCENET Computer Conferences on biogas production, anaerobic digestion and the bioconversion of lignocellulose. Computer conferencing is a network system that links geographically scattered nodes together through the use of home or office computers to a remote control computer (see Chapter 2). Apart from attempting to link up the MIRCENs and organising specialised conferences, MIRCENET has other functions listed in Table 8.2. On the basis of their research and training programmes, the other MIRCENs can be broadly classified as follows. The Biotechnology MIRCENs. In the area of biotechnology, there are 14 MIRCENs in operation (see Table 8.1). These are in Thailand, Egypt, Guatemala, Japan, Argentina, USA, the UK, Canada, Sweden, France, Australia, China, Trinidad and Tobago. In the region of Southeast Asia, the MIRCEN in Bangkok has cooperating laboratories in the Philippines, Indonesia, Singapore, Malaysia and Hong Kong and other institutions in Thailand. It serves the microbiological community in the collection, preservation, identification and distribution of microbial germplasm, and in the promotion of research and training activities directed towards the needs of the region. In the region of the Arab States, the MIRCEN at Ain-Shams University, Cairo, promotes research and training courses on the conservation of microbial cultures and biotechnologies of interest to the region. Table 8.2. Functions of MIRCENET

To help initiate closed computer conferences under defined keys such as microbiology, biological nitrogen fixation, biogas, networking in culture collections. To act as an information source for meetings, reviews, identification services, etc. To provide a platform for discussions of MIRCEN network activities. To provide print-outs and records of MIRCENET entries.

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Through its cooperating MIRCEN laboratory at the University of Khartoum, the MIRCEN has contributed to the establishment of a culture collection in Sudan specialising in fungal taxonomy. The cooperating MIRCEN laboratory at the Institute Agronomique et Veterinaire Hassan II, Rabat, has made commendable progress through projects using different species of yeasts and rhizobia. In the region of Central America and the Caribbean, the MIRCEN (cooperating laboratories in Chile, Columbia, Costa Rica, Dominican Republic, Ecuador, El Salvador, Honduras, Jamaica, Mexico, Nicaragua, Peru, Venezuela) has, in cooperation with the Organization of American States, the Interamerican Development Bank and several other prestigious agencies, pioneered the applications of microbiology, process engineering and fermentation technology in several member states of Central America and the Caribbean. It has set up joint collaborative research projects, the exchange of technical personnel, regional training programmes and the dissemination of scientific information among network institutions. The South American Biotechnology MIRCEN located at Tucuman, Argentina is comprised of a regional network with cooperating laboratories in Brazil, Chile, Bolivia and Peru. It has similar goals to the biotechnology MIRCEN for Central America and the Caribbean. The MIRCENs in the industrialised societies function as a bridge with those in the developing countries. In such a manner, increased cooperation is promoted between the developed and developing countries. Furthermore, a basic structure is set up for eventual twinning at a later date. For example, the Guelph/Waterloo MIRCEN, Canada, with its expertise at the University of Waterloo in biomass conversion technology, microbial biomass protein production and bioreactor design, is of immense benefit to the work of the MIRCENs at Cairo, Guatemala and Tucuman. In a similar manner, the MIRCEN at Bangkok has several collaborative research projects with that at the International Centre of Co-operative Research in Biotechnology, Osaka, Japan. This centre conducts the annual UNESCO International Postgraduate University Course on Microbiology (of 12 months' duration). It also functions as the Japanese point-of-contact for the Southeast Asian regional network of microbiology in the UNESCO Programme for Regional Co-operation in the basic sciences. In the UK there is a MIRCEN network centred upon the Institute for Biotechnological Studies (IBS). In common with other Microbiological

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Resource Centres, the aims of the UK MIRCEN are to promote the utilisation of the microbial gene pool, to promote applied microbiology and biotechnology in the developing countries and to provide a centre for training and advice. The intergovernmental CAB International Mycological Institute (CMI) is the MIRCEN for mycology cooperating with all others in the field world-wide. This organisation, together with the Institute of Horticultural Research (IHR) are the first two organisations to collaborate with the MIRCEN Network, whilst continuing their own activities in mycological and biodeterioration studies, and microbiological pest control, mycorrhizas and mushroom technology respectively. In keeping with the new trends of the expanding frontiers of biotechnological research, a Marine Biotechnology MIRCEN has been established at the University of Maryland. Work presently under way includes the fundamental elucidation of the evolution of genes and the flow of genes through populations in the marine environment. One collaborative study under way is with the Chinese University of Hong Kong and the Shandong College of Oceanography, Qingdao, China. The Biological Nitrogen Fixation (BNF) MIRCENS. In the quest for more

food for their increasing populations, several developing nations have been expanding their agricultural lands into areas which are marginally capable of sustaining productivity and invariably limited by the availability of nitrogen fertilizer. In interaction with other international programmes, modest schemes for the development of biofertilizers or Rhizobium inoculant material, particularly in legume-crop areas of the developing countries, are already operating through the MIRCENs on a level of regional cooperation in Latin America, East Africa and Southeast Asia and the Pacific. In the area of biological nitrogen five MIRCENs are already operating (see Table 8.1). The broad responsibilities of these MIRCENs include collection, identification, maintenance, testing and distribution of rhizobial cultures compatible with crops of the regions. Deployment of local rhizobia inoculant technology and promotion of research are other activities. Advice and guidance are provided in the region to individuals and institutions engaged in rhizobiology research. The BNF MIRCENs play a valuable role in maintaining and distributing efficient cultures of Rhizobium. Nearly 4000 strains are maintained in the MIRCEN collections and about 1750 have been distributed to other organisations (Table 8.3).

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The MIRCEN network is founded on the principle of self-help and mutual cooperation. It concentrates on existing facilities and resources and provides an organisational structure which allows each institution to collaborate as best it can through the following: (1) an exchange of research workers between national and regional institutions; (2) small grants to individual research projects or workers for acquisitions of supplies, spare parts for equipment, or smallscale equipment; (3) participation of senior scientists in specialised symposia in the technically advanced countries in the vicinity of each of (contd. on p . 170) the regions; Table 8.3. Culture collection services of Biological Nitrogen Fixation (BNF) MIRCENs" Holdings of Rhizobium culture collections MIRCEN

Number of strains held

Bambey Beltsville Hawaii Nairobi Porto Alegre Total

50 938 2000 208 650 3846

Cultures distributed by Rhizobium MIRCENs MIRCEN

Number of cultures

Countries of recipient institutions

Bambey Beltsville

8 508

Hawaii Nairobi

200 95

Porto Alegre

943

Gambia, Mali, Yemen Zimbabwe, Nigeria, Yugoslavia, India, Spain, Vietnam, Ireland, UK, Malaysia, Italy, Canada, South Africa, Senegal, Egypt, Poland, Argentina, Turkey, W. Germany, Austria, Australia, New Zealand Global Uganda, Malawi, Tanzania, Mauritius, Sudan, Congo, Zaire, Rwanda Argentina, Chile, Bolivia, Uruguay, Peru, Ecuador, Colombia, Venezuela, El Salvador, Dominican Rep., Mexico, USA, Trinidad, Brazil

1988 figures.

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(4) organisation of short-term intensive training courses and specialised in-depth sub-national or national meetings; (5) production of a newsletter functioning as an outlet for the exchange of research news, publication of research findings and as an attraction for potential participating laboratories. The MIRCENs play a catalytic role in breaching the barrier of geographical isolation and advancing the frontiers of contemporary research in biotechnology through the production of newsletter bulletins, culture collection catalogues and research papers. The publication of MIRCEN News annually, the development of MIRCENET, and the UNESCO MIRCEN Journal of Applied Microbiology and Biotechnology (now incorporated in the World Journal of Microbiology and Biotechnology)

are indications of the gradual emergence of competence and capability of the MIRCENs, and the services they provide on a regional and interregional basis. 8.3

Regional organisation Transnational coordinating mechanisms are being set up throughout the world to bring regional cohesion to culture collection activities and benefit to both the resource centres themselves and their users. Some have been established as committees by culture collections; others have originated as data centres with the secondary effect of stimulating closer working collaboration between the contributing culture collections. They may be contacted for information about microbiological resources, services and general advice. 8.3.1

European Culture Collections' Oranisation (ECCO)

In 1981, at an international conference in Brno, Czechoslovakia, curators of European service culture collections present agreed that a mechanism should be set up to enable meetings to take place on an annual basis for the exchange of ideas and the discussion of common problems. In 1982 the first meeting of ECCO took place at the Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany, and since then meetings have been held in France, the UK, Czechoslovakia, Hungary, the Netherlands and the USSR. Membership has increased steadily as new culture collections are formed or developed to provide a national service. Membership is restricted to collections that provide a service on demand and without restriction, that have as a normal part of their duty the acceptance of cultures, that issue from time to time a list of

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their holdings, and that are in a country with a microbiological society belonging to the Federation of European Microbiological Societies. It was felt that these collections have interests and problems in common that are not shared by research or teaching collections. The Organisation is affiliated to FEMS and an adherent member of the World Federation for Culture Collections. Apart from the exchange of scientific information relating to such topics as taxonomy, identification and preservation procedures, much benefit has been derived from discussions on new developments within culture collections such as acceptance of International Depositary Authority status (see Chapter 6) or the development of computerised systems for the storage, searching and dissemination of culture information (see Chapter 2). In addition, the opportunity to meet on a regular basis has enabled collaborative programmes to be set up between collections from different countries. Discussions are taking place on the possible expansion of ECCO to increase scientific activities and broaden representation. ECCO members have become aware that the services available from the collections are not fully exploited by users. To remedy this they have combined to produce publicity material which may be obtained from the recently established Information Centre for European Culture Collections (ICECC) at Braunschweig, Federal Republic of Germany (see Chapter 2). Information about the holdings and services of ECCO collections is also available from the Organisation's Officers (see Chapter 2) or the Secretary of FEMS. 8.3.2

Regional database systems

A number of coordinating mechanisms based on information centres have been set up, primarily to establish data banks for regional access. These are described in greater detail in Chapter 2. Some, such as the Tropical Data Base in Brazil, the Microbial Information Network Europe (MINE) and the Nordic Register, have been developed initially to provide a centre for information on the culture collections themselves, their services and their holdings. Others, such as the Microbial Culture Information Service (MiCIS), have been set up in areas with well established culture collection systems with the purpose of providing on-line strain databases for searching. Both these kinds of data centres have the secondary effect of encouraging collaboration between culture collections so that the best possible system develops and minimum duplication of effort takes place.

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The proliferation of microbial data centres world-wide reflects the growing need for information on biological materials. It has also led to problems in identifying the most appropriate point of contact for specific information. To overcome this an international system has been established (Microbial Strain Data Network, MSDN), to act as a referral system and communications network to databases able to answer specific enquiries on strain properties (Chapter 2). It seems certain that other systems will be established, and the function of the MSDN thus becomes of increasing importance as the first point of enquiry, directing those seeking information on strain properties to appropriate centres. 8.4

National federations and committees The following countries have established federations or committees for the coordination of culture collection activities: Australia Canada China Czechoslovakia Japan Korea New Zealand Turkey United Kingdom United States of America Information about them and their activities may be obtained through culture collections or microbiological societies within the country or through the World Data Center and the Microbial Strain Data Network (see Chapter 2). Most of these organisations produce newsletters from time to time and further information may be obtained through these publications. Some of the organisations are for culture collections, others are of culture collections and the difference between the two categories is significant. Those that are of culture collections exist primarily to coordinate culture collection activities within the countries (produce common catalogues, rationalise holdings, stabilise funding) and are generally termed Committees rather than Federations; those that are for culture collections have as their prime function the promotion of communication between the collections and their users in industry, research and education. The activities of the latter category concentrate

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more on scientific meetings, workshops and training courses, and the membership includes any microbiologists with an interest in culture collection activities, whether they are working in culture collections or not. The executive boards are deliberately formed of people both from culture collections and from research or teaching laboratories and industry, providing a cross fertilisation of interests, whereas with organisations set up for culture collections the officers and members are drawn from the collections only. The impact of biotechnological input to the Federations has played a valuable part in the development of resource centres to meet the growing needs of industry in this area. A number of international organisations exist for the coordination of activities within different microbiological disciplines, and information about them can be obtained from the International Council for Scientific Unions (Table 8.4). Information on biotechnology is disseminated through the different associations listed in Table 8.5. All these organisations recognise the need for an effective network of microbial resource centres and are active in support of their development. 8.5

Future developments Developments in biotechnology have coincided with extensive advances in computer technology, and throughout the world culture collections have taken advantage of the latter to respond to the increasing demands of the former. It is clear from Chapter 2 that data held in the microbial resource centres is increasingly computerised and it is evident that the biotechnology community can better be served by coordination of these activities. The World Data Center for Collections of Cultures of Microorganisms and the Microbial Strain Data Network

Table 8.4. International scientific organisations

ICSU

IUBS IUMS ICRO

International Council of Scientific Unions 51 Boulevard de Montmorency F-75016 Paris France Telephone: (1) 45-25-03-29 Telex: ICSU 630553 F Electronic mail: TELECOM GOLD 75:DBI0126 International Union of Biological Sciences International Union of Microbiological Societies (International Cell Research Organisation) Panel on Applied Microbiology and Biotechnology

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are important examples of international collaboration in this area, leading to on-line databases and information network systems. The imaginative and successful MIRCEN network will continue to be instrumental in encouraging the establishment and development of culture collection activities in the developing world and linking them to those in industrial nations. Computers will be used increasingly for computer conferencing and electronic mail, leading to greater communication between the collections. This in turn should lead to greater collaborative research and Table 8.5. Biotechnology associations ABA

ABC

ADEBIO

BIA

BIDEC

IBA

IBAC

Australian Biotechnical Association 1 Lorraine Street Hampton Victoria 3188 Australia Association of Biotechnology Companies 1220 L Street NW Suite 615 Washington, DC 20005 USA Association de Biotechnologie 3 rue Massenet F-77300 Fontainebleau France Biolndustry Association 1 Queen's Gate London SW1H 9BT UK C/o Japan Association of Industrial Fermentation 20-5 Shinbashi 5-chome Minato-ku Tokyo 105 Japan Industrial Biotechnology Association 2115 East Jefferson Street Rockville Maryland 20852 USA Industrial Biotechnology Association of Canada Lava University Cite Universitaire Quebec G1K 7P4 Canada

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joint service activities and will minimise unnecessary duplication of effort, consistant with national requirements. In spite of rapid developments in communication systems, the need for the presence of culture collections in all regions of the world will remain because of specialised local needs, regional regulatory requirements, such as those for postal and quarantine purposes, or currency or language reasons. Duplication of important holdings and services is necessary, but can be reduced to an acceptable level by collaborative efforts on the part of individual scientists in the resource centres, the setting up of organisations to coordinate their activities and the use of computers and electronic networking to facilitate communication. A basic core of collaborative mechanisms already exists and can be extended to cover regions of the world or specialist areas of activity not yet coordinated internationally.

INDEX

accessions documentation required, 53-4 policies on, 52-3 accreditation of laboratories, 155 Acinetobacter, Hazard Group, 56 Actinobacillus, Hazard Group, 56 Actinomadura, Hazard Group 56 Actinomyces, Hazard Group, 56 actinomycetes, resource centres holding, 14 activated charcoal, drying on, 67, 68 administration, 49-54 Advisory Committee on Dangerous Pathogens (ACDP), 55f 56 Genetic Manipulation, 59 advisory services, 149 Aeromonas hydrophila, Hazard Group, 56 Africa, resource centres, 4 Agency of Industrial Science and Technology (FRI) (Japan), 9 agricultural bacteriology, resource centres specialising in, 16 Agricultural Research Service Culture Collection (NRRL) (USA), 7,124, 134-7 air-freight regulations, 49, 52 America resource centres, 4-8 see also United States of America (USA) American Type Culture Collection (ATCC), 3, 7, 124, 137 conditions of supply, 60-1

Resources Bioinformatics Department, 45 anaerobic bacteria, drying of, 68 anaerobic conditions, cryopreservation under, 71^4 animal varieties, patent protection for, 100 animals, processes for production of, patent protection for, 100-1 antigen-antibody reactivity, data resource for, 39 Arizona, Hazard Group, 56 Association de Biotechnologie (ADEBIO) (France), 174 Association of Biotechnology Companies (ABC), (USA), 174 Australasia, resource centres, 10-11 Australia IDA, 124 patent system, 106 resource centres, 10, 165, 166 Australian Biotechnical Association (ABA), 176 Australian Government Analytical Laboratory (AGAL), 124, 127 Austria, patent system, 106 Bacillus

Hazard Group, 56 resource centres holding, 6, 7 Bacillus Genetic Stock Center (BGSC) (USA), 7 Bacterial Culture Collection (MCITM) (Belgium), 11 177

178

Index

CAB International Mycological Institute (UK), 125, 131, 165, 168 testing of products, 151 Bacterionemia matruchotti, Hazard Campylobacter, Hazard Group, 56 Group, 56 Canada bacteriophage typing, 86 Bambey (Senegal), rhizobia culture biotechnology association, 176 collection, 4, 165, 167 patent system, 103, 106, 120 Bartonella bacilliformis, Hazard Group, resource centre, 6, 165, 167 56 Cardiobacterium hominis, Hazard Belgium Group, 56 patent system, 106 Caribbean, MIRCEN network, 167 resource centres, 11 Catalogo Nacional de Linhagens, 32 Beltsville Agricultural Research catalogues, 29, 32, 49-51, 156-7 Center (USA), rhizobia culture arrangement of, 27, 50 collection, 8, 165, 167 printed, 50, 156-7 BIDEC (c/o Japan Association of Centraalbureau voor Industrial Fermentation), 174 Schimmelcultures (CBS) biochemical data, 25 (Netherlands), 14, 124, 130 biochemical services, 149-50 Central America, MIRCEN network, biochemical tests, 86, 87 153 Biolndustry Association (BIA) (UK), Centre de Collection de Types 176 Microbiens (CHUV) Biological Nitrogen Fixation (BNF) (Switzerland), 15 MIRCENs, 168, 169 CERDIC (France), 40 see also Rhizobium MIRCENs Chakrabarty (patent law) case, 100, biological testing, 150-3 103 biotechnological applications, 2-3 charts, identification, 86-7 biotechnological inventions chemotaxonomy, 83, 89-91 patent protection, 95-145 China patentability, 98-110 Committee for Culture Collection types, 97-8 of Microorganisms (CCCCM), 8 marine biotechnology studies, 168 biotechnology associations, 176 patent system, 101 biotechnology MIRCENs, 165, 166-8 resource centre, 165 Bordetella, Hazard Group, 56 Chlamydia, Hazard Group, 56 Borrelia, Hazard Group, 56 chromatography, 90 Brazil CIAT Rhizobium Collection information resource, 32 resource centres, 4r-6, 165 (Colombia), 6 British Standards, 152 claims, patent, 112-18 Brucella, Hazard Group, 56 Clostridium, Hazard Group, 56 Budapest Treaty, 101, 104-10 codes of practice, 59 'conversion' of deposits, 141 Colecao de Culturas Adolfo Lutz countries participating, 105 (IAL) (Brazil), 4 deposit requirements, 105-8, Coleccion Espanola de Cultivos Tipo 109-10, 122-43 (CECT) (Spain), 15 depositing procedure, 141-3 Collection of Animal Pathogenic WFCC advice, 102, 161 Microorganisms (CAPM) Bulgaria (Czechoslovakia), 12 IDA, 12 Collection Nationale de Cultures de patent system, 106 Microorganisms (CNCM) resource centres, 11-12 (France), 13, 124, 128 Bulgarian Type Culture (BTCC), 11 Colombia, resource centre, 6

Bacteriological Code of Nomenclature, 82, 88

Index colony variation, 63—4 Committee on Data for Science and Technology (CODATA), 38 Task Groups, 38 composition inventions, 98 computer assisted identification, 84 computer conference facilities, 166, 170 computers records on, 24, 78 see also databases conditions of supply, 60-1 confidentiality, 155 consultancy work, 153 contract services, 150-5 conversion, patents, 127 Corynebacterium, Hazard Group, 57 Coxiella burnetii, Hazard Group, 57 cryopreservation, 68-74 cryoprotectants, 69, 70, 71 Culture Collection of Algae and Protozoa (CCAP) (UK), 125 Culture Collection of Department of Microbiology (KUKENS) (Turkey), 16 Culture Collection of University of Goteborg (CCUG) (Sweden), 15 culture collections catalogues for, 29, 32, 50 fees charged, 18, 51, 157-8 future developments in, 20-1, 173-5 institutional repository, 24 pricing policies used, 18, 51, 157-8 quality control in, 54, 77-8 records for, 53, 78 services available, 148-58 service-supply, 3-19, 24 specialist, 19-20, 29-30 types of, 2, 3-20 see also International Depositary Authorities (IDAs) and resource centres cultures conditions of supply, 60-1 custom preparations of, 154 deposition of, 53 dispatch of, 51-2 growth of, 62-4 maintenance of, 64-77 optimum growth of, 64 prices for, 18, 51, 157-8

179 purity of, 87 supply of, 49 transportation of, 61 cytochrome patterns, 89 Czechoslovak Collection of Microorganisms (CCM), 12, 33 Czechoslovak National Collection of Type Cultures (IEM), 12 dangerous goods, definition of, 52 databases, 22, 36-46, 149 access to, 42, 46-7 identification profiles, 84, 90, 91, 92 information available on, 26 international, 31, 37-46, 172 national, 31, 32, 33, 34r-6 regional, 34, 36-7, 171-2 data-management methods, 24, 78 deep-freezers, storage of cultures in, 68-9 Denmark, patent system, 103, 106 Department of Trade and Industry (DTI) (UK), 35 deposits accessions, 53 patent system, 105-8, 109-10, 121-9 desiccation, preservation by, 66-8 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) (Germany), 13, 129 database, 34, 43 preservation techniques, 67 Directory of Biotechnology Information Resources (DBIR), 45-6 disclosure of invention, 96-7, 111-18 premature, 111 sufficiency of, 103-4 discoveries, distinction from inventions, 97, 102 dispatch arrangements, 51-2 DNA base compositions, 91 DNA fingerprinting, 92 DNA probes, 92-3 documentation accessions, 53-4 specialist collections, 19, 20 drying, preservation by, 66-8

180

Index

Education Committee (WFCC), 44, 142, 148 educational activities, 155-6 Edwardsiella tarda, Hazard Group, 57 Egypt, resource centre, 165, 166-7 Eikenella corrodens, Hazard Group, 57 electronic access, 46-7 electronic mail, 42, 174 electrophoresis, 90-1 Endangered Collections, Committee for (WFCC), 44, 53, 162-3 enquiries, answering of, 20, 30, 46 enrichment procedures, 62-3 Enterobacter aerogenes, Hazard Group, 57 Equipe de Microbiologia do Solo, Instituto de Pesquisas Agronomicas (SEMIA) (Brazil), 6 Erypsipelothrix rhusiopathiae, Hazard Group, 57 Escherichia coli

Genetic Stock Center (USA), 7 Hazard Group, 56 Europe information resources, 32-3, 34, 35-7 resource centres, 11-18, 165 European Collection of Animal Cell Cultures (ECACC), 131 European Culture Collections Organisation (ECCO), 32-3,170-1 European Patent Convention (EPC), 99, 118, 119, 120 European Patent Office (EPO) filing of application, 118, 119 guidelines, 100 as party to Budapest Treaty, 105, 107 patent specification requirements, 112, 113 European Standards, 152 fatty acids, 89, 90 Federation of European Microbiological Societies (FEMS), 171 fees, 18, 51, 143-4 Fermentation Research Institute (FRI), 130 Finland patent system, 106 resource centre, 12

Flavobacterium meningosepticum, Hazard Group, 57 food bacteriology, resource centres specialising in, 16 Food Industry Research and Development Institute (FIRDI) (Taiwan), 8 France IDA, 13, 125 information resources, 38, 40 patent system, 106, 108, 109 resource centre, 13, 165 Francisella tularensis, Hazard Group, 57 freeze-drying NCTC method, 75-7 optimising of, 77 preservation by, 74r-7 protective agents used, 75 freezing techniques, 68-74 containers used, 70, 71 cooling rates used, 69 thawing of cultures, 73 Fundacao Tropical de Pesquisas e Tecnologia 'Andre Tosello' (FTPT) (Brazil), 4, 32 genetically manipulated strains guidelines for use, 59 patent protection, 103 storage, 70 Germany IDA, 13 patent system, 103, 106, 108, 110, 113 resource centres, 13 Gram staining reaction, 86, 87 Guatemala, resource centre, 165 Haemophilus, Hazard Group, 57 Hawaii (USA), rhizobia culture collection, 8, 165, 167 Hazard Groups definition of, 55 listed, 56-8 hazards, data on, 27 Health and Safety at Work etc. Act (1974) (UK), 54, 55 Hungarian National Collection of Medical Bacteria (HNCMB), 13 Hungary IDA, 14

Index patent system, 106, 108 resource centres, 13-14 Hybridoma Data Bank (HDB), 38-40 on-line services, 39

181

Qualidade em Saude (INCQS) (Brazil), 5 Instituto do Patalogia Vegetale (IPV) (Italy), 14 Instituto de Tecnologia de Alimentos, Secao de Leite e Derivados IATA Dangerous Goods Regulations, 52 (ITALSL) (Brazil), 5 identification, 81-93 Secao de Microbiologia (ITALSM) charts for, 86-7 (Brazil), 5 conventional, 83—4 Instituto Zimotecnico (IZ) (Brazil), 5 kits for, 84-5 interdisciplinary information new methods, 83 sources, 23-4 practical aspects, 86-8 International Cell Research probabilistic, 84 Organisation (ICRO), 173 and taxonomy, 82 International Centre of Co-operative traditional methods, 82-3 Research in Biotechnology IMET Kulturensammlung (Japan), 167 (Germany), 13 International Collection of MicroIn Vitro International Inc (IVI) (USA), organisms from Plants (ICMP) 8 (New Zealand), 11 individual collections, 29-30 International Convention for the industrial applicability of invention, Protection of New Varieties of criteria for, 96 Plants (UPOV), 98 Industrial Biotechnology Association International Council of Scientific of Canada (IBAC), 176 Unions (ICSU), 37-8, 173 Industrial Biotechnology Association see also Committee on Data for (IBA) (USA), 176 Science and Technology industrial data, 26 (CODATA) and World infectious substances, definition of, 52 Federation for Culture Information Centre for European Collections (WFCC) Culture Collections (ICECC), 33, International Depositary Authorities 34, 37, 171 (IDAs), 104 information needs, 23-8 listed, 7, 8, 9, 12, 13, 14, 16, 17, 18, information networks, 33, 36-7, 47 124-5 information resources, 22^7 requirements, 122-40 international, 37-46 International Journal of Systematic national, 34-7 Bacteriology, 88 types of, 28-31 international organisations, 37-8, Institute of Applied Microbiology 150-60 (IAM) (Japan), 10 International Union of Biological Institute for Biotechnological Studies Sciences (IUBS), 173 (IBS) (UK), 167 International Union of Institute for Fermentation Osaka, Immunological Societies (IUIS), Culture Collection of (IFO) 38 (Japan), 9 International Union of Institute for Physical and Chemical Microbiological Societies Research (RIKEN) (Japan), 34, (IUMS), 38, 159 35,43 inventions, patent protection for, Instituto Biologico, Segao de Bacterias 95-145 Fitopatogenicas (IBSBF) (Brazil), inventiveness, criteria for, 96 5 Iran, resource centre, 9 Instituto Nacional de Controle de Ireland, patent system, 106

182

Index

isolation procedures, 62 isoprenoid quinones, 90 Italy patent system, 106, 109 resource centre, 14 Japan biotechnology association, 176 Collection of Microorganisms (JCM), 10, 34 Federation for Culture Collections (JFCC), 33 IDA, 9 information resources, 33, 34r-5 patent system, 101, 106, 108, 109, 110, 112, 120, 121 resource centre, 9-10, 165, 167" Kansai Medical School Culture Collection (KMS) (Japan), 9 Kenya, resource centre, 4, 165 'Kingella kingae', Hazard Group, 57 Klebsiella, Hazard Group, 57 Laboratorio de Fisiologia Bacteriana, Fundacao Oswaldo Cruz (LFBFIOCRUZ) (Brazil), 6 Laboratorium voor Microbiologie en Microbiele Genetic (LMG) (Belgium), 11 laboratory accreditation, 155 L-drying, 67-8 Legionellaceae, Hazard Group, 57

Leichenstein, patent system, 106 Leptospira

Hazard Group, 57 resource centres holding, 14 Life Science Research Information Section (LSRIS) (Japan), 34-5, 40 Listeria monocytogenes, Hazard Group, 57

location of strains, 24, 49-50 'living material', definition of, 102 lyophilisation, 74r-7 see also freeze-drying maintenance, advice on, 149 marine bacteria, resource centres holding, 16 Marine Biotechnology MIRCEN, 168 mass spectrometry, 90 media, 63

medical bacteriology isolation procedures used, 62 resource centres specialising in, 4, 5, 6, 10, 11, 12, 13, 15, 16, 17, 18 medically important bacteria, identification of, 93 Microbial Culture Information Service (MiCIS), 34, 35-6, 42,171 Microbial Information Network Europe (MINE), 36-7, 171 Microbial Information System (MICRO-IS), 42 Microbial Strain Data Network (MSDN), 36, 38, 39, 40-3, 172, 173 Central Directory, 40-1 communication media used, 43 databases available, 42 electronic mail service, 42 services, 41 microbiological resource centres (MIRCENs), 33 biotechnology, 165, 166-8 functions of, 164 listed, 4-18 network, 164-70 rhizobia-holding, 165, 168, 169 microorganism definitions of, 101-2 inventions involving, 101-2 MIRCENET, 164, 166 monoclonal antibody techniques, 86 Moraxella, Hazard Group, 57 Morocco, MIRCEN laboratory, 167 morphological data, 26 morphological examination, 86, 87 Mucosplasma pneumoniae, Hazard

Group, 57 multiple colony cultures, 63 Mycdbacterium, Hazard Group, 57 mycolic acids, 89 mycoplasmas, resource centres holding, 11, 17 Nairobi (Kenya), rhizobia culture collection, 4, 165, 167 National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) (Bulgaria), 12, 128 National Collection of Agricultural and Industrial

Index Microorganisms (NCAIM) (Hungary), 14, 130 Food Bacteria (NCFB) (UK), 16 Industrial and Marine Bacteria Ltd (NCMB) (UK), 16, 132-4 Plant Pathogenic Bacteria (NCPPB) (UK), 17 Type Cultures (NCTC) (UK), 17, 135; conditions of supply, 60; freeze-drying method, 75-7; paperwork, 53 yeast cultures (NCYC), 134 national federations/committees, 31, 172-3 National Information System of Laboratory Organisms (NISLO) (Japan), 34 National Library of Medicine (NLM) (USA), information resource, 45, 46 Neisseria

Hazard Group, 57 Reference Laboratory (NRL) (USA), 8 resource centres holding, 8 Netherlands IDA, 14, 125 patent system, 105,107,109,112,120 resource centres, 14-15 New Zealand patent system, 107 Reference Culture Collection (NHI), 11 resource centres, 11 NIFTAL Rhizobium Germplasm Resource (TAL) (USA), 8 nitrogen, liquid, storage in, 69-74 disadvantages of, 74 Nocardia brasiliensis, Hazard Group, 57 NODAI Research Institute Culture Collection (Japan), 33 'Noguchia granulosis', Hazard Group, 57 nomenclature new species, 88 rules for, 82 Nordic Register of Microbiological Culture Collections, 37, 171 Norway, patent system, 107 novelty of invention, criteria for, 96, 111

183 nucleic acids base sequence similarities, 91-2 identification by, 91-3 observation search strategy, 25 Occupational Safety and Health Act (1970) (USA), 54 on-line services, 35, 36, 39, 41, 45, 47 Organization for Economic Cooperation and Development (OECD), patent protection questionnaire, 103 packet switching service (PSS) transmission, 47 Pasteurella, Hazard Group, 57 patent application filing, 118-19 Budapest Treaty requirements, 104-10, 122-9 claims, 112-18 'converted7 deposits, 127 deposit requirements, 105-8, 109-10, 121-9 disclosure requirements, 96-7, 103-4, 111-8 national deposit requirements, 105-8 new (replacement) deposits, 140-1 office procedures, 119-21 practical considerations, 110-45 principles, 95-7 protection by, 95-145 release of samples, 108-9 samples, 108-9, 143-5 technical description, 111-12 patent agent/attorney, 110-11, 119, 120, 143 Patent Procedures Committee (WFCC), 44, 102, 151 patentability biotechnological inventions, 98-110 criteria for, 96-7, 100 exclusions for, 97, 99, 100-1 pathogenicity, data on, 27 pathogens categorisation of, 55-9 identification of, 93 precautions when freeze-drying, 77 storage of, 70

184

Index

peptidoglycans, 89 Persian Type Culture Collection (PTCC) (Iran), 9 Phabagen Collection (Netherlands), 15 phenotypic classification, 82 Philippine Type Culture Collection (PTCC), 10 physiological data, 25-6 physiological tests, 86, 87 plant pathogenic bacteria, resource centres holding, 5, 11, 17 plant variety rights, 98-100 plants, processes for production of, patent protection for, 100-1 plasmids, resource centres holding, 11, 12, 15, 17 Plesiomonas shigelloides, Hazard

Group, 58 Polish Collection of Microorganisms (PCM), 15 Porto Alegre (Brazil), rhizobia culture collection, 6, 165, 167 Portugal, patent system, 107 postal regulations, 52 WFCC Committee dealing with, 44, 161 pre-preservation culture, 64 preservation techniques, 66-77 advice on, 149 pricing policies, 18, 51, 143-4 'prior art', 96 'priority date', 96, 118 'priority document', 118 probabilistic identification, 84 process inventions, 98 product inventions, 97 proteins, electrophoresis of, 90-1 Proteus, Hazard Group, 58 Providencia, Hazard Group, 58 Pseudomonas pseudomallei, Hazard

Group, 58 Public Health Laboratory Service (PHLS) (UK), Code of Practice, 59 publications culture collection, 156-7 patent, 109 Publicity Committee (WFCC), 163 publicity material culture collections, 157 ECCO, 171

purity check plates, retention of, 63 pyrolysis gas—liquid chromatography, 90 quality control, 54, 77-8 Quarantine and Postal Regulations Committee (WFCC), 44, 161 records, culture collections, 53, 78 referral systems, 36, 172 regional database systems, 34, 36-7, 171-2 regional organisations, 170-2 regulations airfreight, 49, 52 data on, 28 postal, 52 research and development work, 153-4 resource, meaning of term, 1-2 resource centres, 1-21 future development of, 20-1,173-5 listed, 4-18, 165 organisation of, 160-75 types of, 3-20 restriction endonucleases, 92 RhizobiumMIRCENs, 4, 6, 8,17,165, 167, 168, 169 Rickettsia, Hazard Group, 58 RIKEN (Japan), 34, 35, 43, 164 risk-assessment data, 27 Rochalimaea quintana, Hazard Group, 58 Rothamsted Rhizobium Collection (RCR) (UK), 17 safe deposits, 148-9 safety, 54-61 Salmonella, Hazard Group, 58 Salmonella Genetic Stock Centre (LSCC) (Canada), 6 searching, 26, 29 security of records, 78 selective media, 63 Senegal, resource centre, 4, 165 serological classification, defects of, 85-6 Serratia, Hazard Group, 58 service-supply collections, 3-19, 24 catalogues, 28, 32, 50 pricing policies, 18, 51, 157-8 Shigella, Hazard Group, 58

Index South America MIRCEN network, 157 resource centres, 4r-6, 165, 167 Spain patent system, 107 resource centre, 15 specialist collections, 19-20, 29-30 spin-freezing, 76 staff, 20-1 standardised tests, identification by, 87-8 standards, testing, 138-9 Standards Committee (WFCC), 163 Staphylococcus aureus, Hazard Group, 58 storage techniques, 66-77 strain data, 25-7 strain data compendia, 30 strain data networks, 149 strains accessing of, 25 locating of, 24, 49-50 selection advice on, 149 uniqueness of, 53-4 Streptobacillus moniliformis, Hazard

Group, 58 Streptococcus, Hazard Group, 58 Streptomyces, resource centres holding, 7, 14 subculturing, 64-6 containers used, 65 disadvantages of, 66 Sudan, MIRCEN Laboratory, 167 supply of cultures, 49 suspensions checking of, 64 preparation for cryopreservation, 71, 73 Sweden patent system, 107, 109 resource centre, 15, 165, 166 Switzerland patent system, 107, 108 resource centre, 15 Taiwan, resource centre, 8 taxonomic data, 27-8 taxonomy, and identification, 82 telecommunications, 46-7 telephone enquiries, 20 test results, evaluation of, 88

185 Thailand, resource centres, 10, 165, 166 theorems, distinction from inventions, 97 tissue cultures, patentability of, 101 TISTR Culture Collection (Thailand), 10 training computers in microbiology, 42 culture collection expertise, 21, 155-6 transportation precautions for, 61 recommendations for, 161 Treponema, Hazard Group, 58 Trinidad and Tobago, resource centre, 165 Turkey, resource centre, 16 ultra-low-temperature preservation, 68-74 Union of Socialist Soviet Republics (USSR) IDAs, 18 patent system, 100, 107, 110 resource centres, 18 United Kingdom (UK) biotechnology association, 176 IDAs, 16, 17, 125 information resource in, 35-6, 43 patent system, 101, 103, 107, 108, 112, 120 resource centres, 16-17, 165, 167-8 testing standards, 152 United States of America (USA) biotechnology associations, 176 IDAs, 7, 8, 124 information resources, 39, 45-6 patent system, 101, 103, 107, 108, 110, 111, 112, 120, 144 resourse centres, 7-8, 165, 168 testing standards, 153 Universidade Federal de Pernambuco, Departmento de Antibioticos (UFPEDA) (Brazil), 6 Universidade Federal do Rio de Janeiro, Instituto de Microbiologia (UFRJIM) (Brazil), 6

186

Index

University of Queensland, Department of Microbiology (UQM) (Australia), 10, 165 USDA Rhizobium Culture Collection (BRCC), 8 use, methods of, as inventions, 98 USSR Collection of Microorganisms (RIA), 18 USSR Collection of Microorganisms (VKM), 18 utility of invention, criteria for, 96 vacuum drying, 66-7 veterinary bacteriology, resource centres specialising in, 12,13,16, 17 viability checking of, 77-8 estimation of, 73-4 Vibrio, Hazard Group, 58 virology, resource centres specialising in, 12 VTT Collection of Industrial Microorganisms (Finland), 12 Wellcome Bacterial Collection (WRL) (UK), 17 WHO/FAO Collaborating Centre for Reference and Research in Leptospirosis (ITH)

(Netherlands), 14 World Data Center for Collections of Microorganisms (WDC), 42, 43-4, 163-4, 166, 173 communication media used, 44 location, 43, 164 tasks, 43 World Federation for Culture Collections (WFCC), xi-xii, 44, 160-4 committees, 161-3 data centres, 163-4 Education Committee, 44, 156, 162 Endangered Collections, Committee on, 44, 53, 162-3 information initiatives, 40, 43 Patents Committee, 44, 102, 161 Postal and Quarantine Committee, 44, 161 Publicity Committee, 163 Standards Committee, 163 World Health Organization, guidelines accepted by, 59 World Intellectual Property Organization (WIPO), 102, 161 patent guidelines, 102, 122-3 Yersinia, Hazard Group, 58